Treatment of stroke and other acute neural degenerative disorders via intranasal administration of umbilical cord-derived cells

ABSTRACT

This invention relates to methods of treating stroke by intranasal administration of umbilical cord tissue-derived cells, which are isolated from mammalian umbilical cord tissue substantially free of blood or expanded in culture from a cell isolated from mammalian umbilical cord tissue substantially free of blood, are capable of self-renewal and expansion in culture, and do not produce CD117 and/or telomerase. The methods of the invention regenerate, repair and improve neural tissue and improve behavior and neurological function in stroke patients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No. 12/697,081, filed Jan. 29, 2010, which is a divisional of U.S. application Ser. No. 11/315,898, filed Dec. 22, 2005, now U.S. Pat. No. 7,875,272, issued Jan. 25, 2011, which claims the benefit of U.S. Provisional Application 60/638,966, filed Dec. 23, 2004, and is a continuation-in-part of U.S. application Ser. No. 10/877,269, filed Jun. 25, 2004, now U.S. Pat. No. 7,524,489, issued Apr. 28, 2009, which claims the benefit of U.S. Provisional Application No. 60/483,264, filed Jun. 27, 2003, the entire contents of all of which are incorporated by reference herein. Other related applications include the following commonly-owned, co-pending applications, the entire contents of which are incorporated by reference herein: U.S. application Ser. No. 10/877,012, filed Jun. 25, 2004, now U.S. Pat. No. 7,510,873, issued Mar. 31, 2009; U.S. application Ser. No. 10/877,446, filed Jun. 25, 2004; U.S. application Ser. No. 10/877,445, filed Jun. 25, 2004; U.S. application Ser. No. 10/877,541, filed Jun. 25, 2004, now U.S. Pat. No. 7,413,734, issued Aug. 19, 2008; U.S. application Ser. No. 10/877,009, filed Jun. 25, 2004, now U.S. Pat. No. 7,560,276, issued Jul. 14, 2009; U.S. application Ser. No. 10/876,998, filed Jun. 25, 2004; U.S. Provisional Application No. 60/555,908, filed Mar. 24, 2004; and U.S. patent application Ser. No. 11/315,943, filed Dec. 22, 2005, now U.S. Pat. No. 7,875,273, issued Jan. 25, 2011.

FIELD OF THE INVENTION

This invention relates to the field of cell based or regenerative therapy for stroke patients. In particular, the invention provides cells derived from umbilical tissue having the capability to regenerate, repair, and improve neural tissue and to improve behavior and neurological function in stroke patients or patients suffering from other forms of acute neurological stress or damage, and methods for using such umbilical tissue-derived cells to regenerate, repair, and improve neural tissue and to improve behavior and neurological function in such individuals.

BACKGROUND OF THE INVENTION

Various patents and other publications are referred to throughout the specification. Each of these publications is incorporated by reference herein, in its entirety.

Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety.

In the United States, about 795,000 people experience new or recurrent stroke. About 61,000 of these are first attacks and 185,000 are recurrent attacks (American Heart Association, Heart Disease and Stroke Statistics Update 2004). Preliminary data from 2006 indicate that stroke accounted for about one of every 18 death in the United States (Heron et al. 2008. National Vital Statistics Reports 56:16). On average, every 40 seconds someone in the United States has a stroke. Preliminary stroke mortality in 2006 was 137,265 and the preliminary death rate was 43.6%.

Two of the most common strokes are ischemic and hemorrhagic strokes. In ischemic strokes, a lack of oxygen flow to the brain can result in apoptosis and necrosis of brain tissue leading to an infarction. Similar to cardiovascular ischemia, brain ischemia can be caused by various factors such as blood clots, thrombosis, embolism, blockage by atherosclerotic plaques, or other obstructions in the vasculature. Hypercholesterolemia, hypertension, diabetes, and obesity, among other things, have been identified as risk factors for ischemic strokes. Ischemic strokes are a leading cause of death of human beings worldwide. A ruptured blood vessel in the brain typically causes hemorrhagic strokes, which account for between about 10 and 20 percent of all strokes. The rupture causes bleeding into the brain, where the accumulating blood can damage surrounding neural tissues.

The stroke episode, regardless of its cause, results in neural cell death, especially at the location of the obstruction or hemorrhage. In addition, biochemical reactions that occur subsequent to the stroke episode in the vasculature may lead to edema, hemorrhagic transformation, and a further compromise in neurological tissue. The neurological damage and neuron cell death that result from a stroke can be physically and mentally debilitating to an individual. Among other things, a stroke can result in problems with emotional control, awareness, sensory perception, speech, hearing, vision, cognition, movement and mobility, and can cause paralysis.

The current treatment for ischemic stroke, tissue plasminogen activator (TPA), while efficacious, is only effective if it is administered within three hours of the ischemic event. Therefore, treatment with TPA remains inadequate for patients who are unable to be diagnosed and treated within that time frame. TPA is also not appropriate for patients who suffer a hemorrhagic stroke. Considering the limitations of TPA and the high mortality rate of stroke patients, there is a clear, unmet need to reduce mortality rates, restore neural function and extend the current window of therapeutic opportunity. Effective treatments will have to have a wide window of therapeutic opportunity, address the neurological damage associated with stroke and reduce disabilities that severely reduce quality of life for stroke patients. Cell therapy, a novel approach to treating stroke, may offer significant health benefits to patients.

Neurological damage and neurodegenerative diseases were long thought to be irreversible because of the inability of neurons and other cells of the nervous system to grow in the adult body. However, the recent advent of stem cell-based therapy for tissue repair and regeneration provides promising treatments for a number of neurodegenerative pathologies and other neurological disorders. Stem cells are capable of self-renewal and differentiation to generate a variety of mature neural cell lineages. Transplantation of such cells can be utilized as a clinical tool for reconstituting a target tissue, thereby restoring physiologic and anatomic functionality. The application of stem cell technology is wide-ranging, including tissue engineering, gene therapy delivery, and cell therapeutics, i.e., delivery of biotherapeutic agents to a target location via exogenously supplied living cells or cellular components that produce or contain those agents (For a review, see Tresco, P. A. et al., (2000) Advanced Drug Delivery Reviews 42:2-37). The identification of stem cells has stimulated research aimed at the selective generation of specific cell types for regenerative medicine.

Cell transplantation to replace lost neurons is a new approach to the treatment of the neurological damage that results from a stroke. One obstacle to realization of the therapeutic potential of stem cell technology has been the difficulty of obtaining sufficient numbers of stem cells. Embryonic, or fetal tissue, is one source of stem cells. Embryonic stem and progenitor cells have been isolated from a number of mammalian species, including humans, and several such cell types have been shown capable of self-renewal and expansion, as well differentiation into all neurological cell lineages (Svendsen, C. V. et al. (1997) Exp. Neurol. 148:135-146; Freed, C. R. et al. (2001) New Engl J. Med. 344(10):-719; Burnstein, R. M. et al. (2003) Int. J. Biochem. Cell Biol. 36:702-713; Zhang, S-C. et al. (2001) Nat. Biotechnol. 19:1129-1133; Reubinoff, B. E. et al. (2001) Nat. Biotechnol. 19:1134-1140; Björklund, L. M. et al. (2002) Proc. Natl. Acad. Sci. USA 99(4):2344-2349). However, the derivation of stem cells from embryonic or fetal sources has raised many ethical and moral issues that are desirable to avoid by identifying other sources of multipotent or pluripotent cells.

Stem cells with neural potency also have been isolated from adult tissues. Neural stem cells exist in the developing brain and in the adult nervous system. These cells can undergo expansion and can differentiate into neurons, astrocytes and oligodendrocytes. However, adult neural stem cells are rare, as well as being obtainable only by invasive procedures, and may have a more limited ability to expand in culture than do embryonic stem cells.

Other adult tissue may also yield progenitor cells useful for cell-based neural therapy. For instance, it has been reported recently that adult stem cells derived from bone marrow and skin can be expanded in culture and give rise to multiple lineages, including some neural lineages (Azizi, S. A. et al. (1998) Proc. Natl. Acad. Sci. USA 95:3908-3913; Li, Y. et al. (2001) Neurosci. Lett. 315:67-70). Intrastriatal and intranigral grafting of other cell types, such as neurons derived from human teratocarcinoma, and human umbilical cord blood mononuclear cells have also been tested in animal models of Parkinson's disease (Baker, K. A. et al. (2000) Exp. Neurol. 162:350-360; Ende, N. and R. Chen (2002) J. Med. 33(1-4):173-180).

Postpartum tissues, such as the umbilical cord and placenta, have generated interest as an alternative source of stem cells. For example, methods for recovery of stem cells by perfusion of the placenta or collection from umbilical cord blood or tissue have been described. A limitation of stem cell procurement from these methods has been an inadequate volume of cord blood or quantity of cells obtained, as well as heterogeneity in, or lack of characterization of, the populations of cells obtained from those sources.

Thus, alternative sources of adequate supplies of cells having the ability to differentiate into an array of neural cell lineages remain in great demand. Further, no satisfactory method exists to repair the neural, neurological, and behavioral damage caused by strokes. Strokes can affect the motor system, rendering the patient with symptoms of hemiparesis or paralysis. Thus, a reliable, well-characterized and plentiful supply of substantially homogeneous populations of cells having the ability to differentiate into an array of neural cell lineages would be an advantage in a variety of diagnostic and therapeutic applications for neural repair, regeneration, and improvement, as well as applications for improvements in behavior and neurological function, particularly in stroke patients.

SUMMARY OF THE INVENTION

The problems presented are solved by the compositions, methods and kits of the illustrative embodiments described herein. This invention provides compositions and methods applicable to cell-based or regenerative therapy for neurological diseases and disorders (such as e.g. stroke). In particular, the invention features pharmaceutical compositions, devices and methods for the regeneration or repair of neural tissue using umbilical cord tissue-derived cells.

One aspect of the invention features an isolated umbilical cord tissue-derived cell substantially free of blood, wherein the cell is capable of self-renewal and expansion in culture and has the potential to differentiate into a cell of a neural phenotype; wherein the cell requires L-valine for growth and is capable of growth in at least about 5% oxygen. This cell further comprises one or more of the following characteristics: (a) potential for at least about 40 doublings in culture; (b) attachment and expansion on a coated or uncoated tissue culture vessel, wherein the coated tissue culture vessel comprises a coating of gelatin, laminin, collagen, polyornithine, vitronectin, or fibronectin; (c) production of at least one of tissue factor, vimentin, and alpha-smooth muscle actin; (d) production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C; (e) lack of production of at least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flow cytometry; (f) expression of a gene, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, is increased for at least one of a gene encoding: interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; tumor necrosis factor, alpha-induced protein 3; (g) expression of a gene, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, is reduced for at least one of a gene encoding: short stature homeobox 2; heat shock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1); elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeo box 2 (growth arrest-specific homeo box); sine oculis homeobox homolog 1 (Drosophila); crystallin, alpha B; disheveled associated activator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogen binding protein); src homology three (SH3) and cysteine rich domain; cholesterol 25-hydroxylase; runt-related transcription factor 3; interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein 5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2; neuroblastoma, suppression of tumorigenicity 1; insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1; potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4; integrin, beta 7; transcriptional co-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1; early growth response 3; distal-less homeo box 5; hypothetical protein F1120373; aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan; transcriptional co-activator with PDZ-binding motif (TAZ); fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE binding protein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle); similar to neuralin 1; B cell translocation gene 1; hypothetical protein FLJ23191; and DKFZp586L151; (h) secretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1; and (i) lack of secretion of at least one of TGF-beta2, ANG2, PDGFbb, MIP1b, 1309, MDC, and VEGF, as detected by ELISA.

In specific embodiments, the umbilicus-derived cell has all identifying features of any one of: cell type UMB 022803 (P7) (ATCC Accession No. PTA-6067); or cell type UMB 022803 (P17) (ATCC Accession No. PTA-6068).

In certain embodiments, umbilical cord tissue-derived cells are isolated in the presence of one or more enzyme activities comprising metalloprotease activity, mucolytic activity and neutral protease activity. Preferably, the cells have a normal karyotype, which is maintained as the cells are passaged in culture. In preferred embodiments, the umbilical-derived cells comprise each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-A,B,C and do not comprise any of CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.

Another aspect of the invention features a cell population comprising the umbilical cord tissue-derived cells as described above. In one embodiment, the population is a substantially homogeneous population of the umbilical cord tissue-derived cells. In a specific embodiment, the population comprises a clonal cell line of the umbilical cord tissue-derived cells. In another embodiment, the population is a heterogeneous population comprising the umbilical cord tissue-derived cells and at least one other cell type. In certain embodiments, the other cell type is an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell or other multipotent or pluripotent stem cell. In other embodiments, the cell population is cultured in contact with one or more factors that stimulate stem cell differentiation toward a neural lineage.

Also featured in accordance with the present invention is a cell lysate prepared from umbilical cord tissue-derived cells. The cell lysate may be separated into a membrane enriched fraction and a soluble cell fraction. The invention also features an extracellular matrix produced by the umbilical cord tissue-derived cells, as well as a conditioned medium in which the cells have been grown.

Another aspect of the invention features a method of treating a patient having a neurodegenerative condition (e.g. stroke), the method comprising administering to the patient umbilical cord tissue-derived cells as described above, in an amount effective to treat the neurodegenerative condition (e.g. stroke). In certain embodiments, the neurodegenerative condition is an acute neurodegenerative condition, such as a brain trauma, spinal cord trauma or peripheral nerve trauma. In other embodiments, it is a chronic or progressive neurodegenerative condition, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, tumor, multiple sclerosis or chronic peripheral nerve injury.

In one embodiment, the umbilical cord tissue-derived cells are induced in vitro to differentiate into a neural lineage cells prior to administration. In another embodiment, the cells are genetically engineered to produce a gene product that promotes treatment of the neurodegenerative condition.

In certain embodiments, the cells are administered with at least one other cell type, such as an astrocyte, oligodendrocyte, neuron, neural progenitor, genetically engineered cell, neural stem cell or other multipotent or pluripotent stem cell. In these embodiments, the other cell type can be administered simultaneously with, or before, or after, the umbilical cord tissue-derived cells. Likewise, in these or other embodiments, the cells are administered with at least one other agent, such as a drug for neural therapy, or another beneficial adjunctive agent such as an anti-inflammatory agent, anti-apoptotic agents, antioxidant or growth factor. In these embodiments, the other agent can be administered simultaneously with, or before, or after, the umbilical cord tissue-derived cells.

In certain embodiments, the cells are administered at a pre-determined site in the central or peripheral nervous system of the patient, where they may exert one or more effects, including trophic effect on the nervous system of the patient. They can be administered by injection or infusion, or encapsulated within an implantable device, or by implantation of a matrix or scaffold containing the cells.

In one embodiment, the acute neurodegenerative condition results from ischemic stroke while in another embodiment the condition results from hemorrhagic stroke.

Accordingly, one embodiment of the invention is a method of treating stroke in a patient comprising intranasally administering to the patient umbilical cord tissue-derived cells in an amount effective to treat stroke, whereby the cells (1) are isolated from mammalian umbilical cord tissue substantially free of blood or are expanded in culture from a cell isolated from mammalian umbilical cord tissue substantially free of blood, (2) are capable of self-renewal and expansion in culture, and (3) do not produce CD117. In one embodiment, the umbilical cord tissue-derived cells do not express hTERT or telomerase. In another embodiment, the umbilical cord-tissue derived cells further have one or more of the following characteristics:

(a) express each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C;

(b) do not express any of CD31, CD34, CD45, CD80, CD86, CD 117, CD141, CD178, B7-H2, HLA-G, or HLA-DR,DP,DQ;

(c) secrete at least one of MCP-1, IL-6, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, and TIMP1;

(d) do not secrete at least one of TGF-beta2, ANG2, PDGFbb, MIP1a, 1309, MDC, and VEGF, as detected by ELISA; and

(e) increased expression of interleukin-8; reticulon 1; and chemokine receptor ligand (C-X-C motif) ligand 3, relative to that of a human cell, which is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell.

In one embodiment, the stroke is ischemic stroke. In another embodiment, the stroke is hemorrhagic stroke. Optionally, the method further comprises treating the patient with hyaluronidase prior to intranasally administering the cells to the patient. In another embodiment, the method further comprises administering an immunosuppressive agent such as e.g. cyclosporine. The cells may be administered with at least one other cell type of an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell, genetically engineered cell, or other multipotent or pluripotent stem cell. The at least one other cell type is administered simultaneously with, or before, or after, the umbilical-derived cells. The umbilical-derived cells may also be genetically engineered to produce a gene product that promotes treatment of stroke.

In one embodiment, the umbilical-derived cells exert a trophic effect on the nervous system of the patient. In another embodiment, the umbilical cord tissue-derived cells are induced in vitro to differentiate into a neural cell line prior to administration.

Yet another embodiment of the invention is a method of treating stroke in a patient comprising intranasally administering to the patient a pharmaceutical composition comprising umbilical cord tissue-derived cells in an amount effective to treat stroke, wherein the cells (1) are isolated from mammalian umbilical cord tissue substantially free of blood or are expanded in culture from a cell isolated from mammalian umbilical cord tissue substantially free of blood, (2) are capable of self-renewal and expansion in culture, and (3) do not produce CD117. In one embodiment, the umbilical cord tissue-derived cells do not express hTERT or telomerase. In another embodiment, the umbilical cord-tissue derived cells further have one or more of the following characteristics:

(a) express each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C;

(b) do not express any of CD31, CD34, CD45, CD80, CD86, CD 117, CD141, CD178, B7-H2, HLA-G, or HLA-DR,DP,DQ;

(c) secrete at least one of MCP-1, IL-6, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, and TIMP1;

(d) do not secrete at least one of TGF-beta2, ANG2, PDGFbb, MIP1a, 1309, MDC, and VEGF, as detected by ELISA; and

(e) increased expression of interleukin-8; reticulon 1; and chemokine receptor ligand (C-X-C motif) ligand 3, relative to that of a human cell, which is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell.

The stroke may be ischemic or hemorrhagic stroke. Optionally, the method further comprises treating the patient with hyaluronidase prior to intranasally administering the pharmaceutical compositions to the patients. The pharmaceutical composition may further contain hyaluronidase or an immunosuppressive agent (such as e.g. cyclosporine). In certain embodiments, the pharmaceutical composition is administered as a spray, aerosol, gel, solution, emulsion, or suspension. The pharmaceutical composition may be administered directly to the upper airways such as the paranasal sinuses. In one embodiment, the pharmaceutical composition is administered via a microcatheter. Optionally, the pharmaceutical composition further comprises at least one other cell type such as e.g. an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell, genetically engineered cell, or other multipotent or pluripotent stem cell. In one embodiment, the umbilical cord tissue-derived cells are genetically engineered to produce a gene product that promotes treatment of stroke. In one embodiment, the pharmaceutical composition exerts a trophic effect on the nervous system of the patient. In another embodiment, the umbilical cord tissue-derived cells are induced in vitro to differentiate into a neural cell line prior to administration.

Another aspect of the invention features a pharmaceutical composition for treating a patient having a neurodegenerative condition comprising a pharmaceutically acceptable carrier and the umbilical cord tissue-derived cells described above. The neurodegenerative condition to be treated may be an acute neurodegenerative condition, or it may be a chronic or progressive condition.

In certain embodiments, the pharmaceutical composition comprises cells that have been induced in vitro to differentiate into a neural lineage cells prior to formulation of the composition, or cells that have been genetically engineered to produce a gene product that promotes treatment of the neurodegenerative condition.

In certain embodiments, the pharmaceutical composition comprises at least one other cell type, such as astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell or other multipotent or pluripotent stem cell. In these or other embodiments, the pharmaceutical composition comprises at least one other agent, such as a drug for neural therapy, or another beneficial adjunctive agent such as an anti-inflammatory agent, anti-apoptotic agents, antioxidant or growth factor.

In certain embodiments, the pharmaceutical composition is formulated for administration by injection or infusion. Alternatively, it may comprise an implantable device in which the cells are encapsulated, or a matrix or scaffold containing the cells.

According to yet another aspect of the invention, a kit is provided for treating a patient having a neurodegenerative condition. The kit comprises a pharmaceutically acceptable carrier, a population of the above-described umbilical cord tissue-derived cells and instructions for using the kit in a method of treating the patient. The kit may further comprise at least one reagent and instructions for culturing the umbilical cord tissue-derived cells. It may also comprise a population of at least one other cell type, or at least one other agent for treating a neurodegenerative condition.

According to another aspect of the invention, a method is provided for treating a patient having a neurodegenerative condition, which comprises administering to the patient a preparation made from the above-described umbilical cord tissue-derived cells. Such a preparation may comprise a cell lysate (or fraction thereof) of the umbilical cord tissue-derived cells, an extracellular matrix of the umbilical cord tissue-derived cells, or a conditioned medium in which the umbilical cord tissue-derived cells were grown. In another aspect, the invention features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a preparation made from the umbilical cord tissue-derived cells, which may be a cell lysate (or fraction thereof) of the umbilical cord tissue-derived cells, an extracellular matrix of the umbilical cord tissue-derived cells or a conditioned medium in which the umbilical cord tissue-derived cells were grown. Kits for practicing this aspect of the invention are also provided. These may include the one or more of a pharmaceutically acceptable carrier or other agent or reagent, one or more of a cell lysate or fraction thereof, an extracellular matrix or a conditioned medium from the umbilical cord tissue-derived cells, and instructions for use of the kit components.

Other features and advantages of the invention will be apparent from the detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

FIG. 1. Graph showing results of Modified Neurological Severity Score (mNSS) Test following middle cerebral artery occlusion (MCAo) in rats (PPC=postpartum-derived cells; MSC=mesenchymal stem cells).

FIG. 2. Graph showing results of Foot-Fault Test following middle cerebral artery occlusion (MCAo) in rats (PPC=postpartum-derived cells; MSC=mesenchymal stem cells).

FIG. 3. Graph showing results of Adhesive Removal Test following middle cerebral artery occlusion (MCAo) in rats (PPC=postpartum-derived cells; MSC=mesenchymal stem cells).

FIG. 4. Graph showing results of Corner Test following middle cerebral artery occlusion (MCAo) in rats (PPC=postpartum-derived cells; MSC=mesenchymal stem cells).

FIG. 5. A cornal brain section showing the areas labeled 1 to 8 used for the microscopic analysis for the studies in Examples 23 and 24. With reference to FIG. 5, IBZ is the ischemic boundary zone.

FIGS. 6, 7 and 8. Graphs showing Modified Neurological Severity Score (mNSS) test results following MCAo in rats for the various routes of hUTC administration in Example 23. FIG. 6A shows mNSS for IA route of administration. FIG. 6B shows mNSS for intravenous (IV) route of administration. FIG. 7A shows mNSS for intracisterna magna (ICM) route of administration. FIG. 7B shows mNSS for intrathecal (IT) route of administration. FIG. 8 shows mNSS for intracerebral (IC) route of administration. In FIGS. 6, 7 and 8, data are presented as mean±standard error (SE). hUTC are Cell A and PBS control is Cell B.

FIGS. 9, 10 and 11. Graphs showing Adhesive Patch Removal test results following MCAo in rats for the various routes of hUTC administration in Example 23. FIG. 9A shows the Adhesive Patch Removal test results for IA route of administration. FIG. 9B the Adhesive Patch Removal test results for IV route of administration. FIG. 10A shows the Adhesive Patch Removal test results for ICM route of administration. FIG. 10B the Adhesive Patch Removal test results for IT route of administration. FIG. 11 shows the Adhesive Patch Removal test results for IC route of administration. Data presented as mean±SE. Each data point represents an average of three timed trials. hUTC (cell A) PBS control (Cell B).

FIGS. 12, 13 and 14. Graphs showing Foot-Fault test results following MCAo in rats for the various routes of hUTC administration in Example 23. FIG. 12A shows the Foot-Fault test results for IA route of administration. FIG. 12B shows the Foot-Fault test results for IV route of administration. FIG. 13A shows the Foot-Fault test results for ICM route of administration. FIG. 14B shows the foot-fault test results for IT route of administration.

FIG. 15. Graph showing plotted CT values for human genomic DNA standard curve generation following MCAo in Example 23.

FIG. 16. Graph showing serum IG levels 7, 14, 28 and 60 days post hUTC injection following MCAo in Example 23. FIG. 16 shows experimental animals that received hUTC (top lines) via intraarterial (A), intravenous (B), intrathecal (C), intracerebral (D), or infra-cisterna magna (E) route of administration compared to animals that received PBS vehicle control (bottom lines). For the results shown in FIG. 16, two animals were missing baseline samples with the CM group for delta MFI calculation. Delta MFI was calculated from the average MFI of baseline samples of these two animals.

FIG. 17. Graph showing neurological functional outcomes following MCAo in rats. FIG. 17 shows the neurological function outcomes following MCAo in rats for hUTC administration with or without cyclosporine (CsA) at day 1 (see Example 24). FIG. 17A shows the results of the Adhesive Removal Test. FIG. 17B shows the results of the Foot-Fault Test and FIG. 17C shows the results of the mNSS test. *p<0.05 for (a) hUTC vs. Vehicle, (b) hUTC+CsA and Vehicle+CsA, (c) hUTC vs. hUTC+CSA (n=8/group). The statistic differences were based on the global test on all three tests.

FIG. 18. Graph showing neurological functional outcomes following MCAo in rats. FIG. 18 shows the neurological function outcomes for hUTC administration at 15 days after MCAo (see Example 24). FIG. 18A shows the results of the Adhesive Removal Test. FIG. 18B shows the results of the Foot-Fault Test and FIG. 18C shows the results of the mNSS test. *p<0.05 for hUTC vs. vehicle (n=8/group). The statistic differences were based on the global test on all three tests.

FIG. 19. Graph showing neurological functional outcomes following MCAo in rats. FIG. 19 shows the neurological function outcomes for hUTC administration at 30 days after MCAo (see Example 24). FIG. 19A shows the results of the Adhesive Removal Test. FIG. 19B shows the results of the Foot-Fault Test and FIG. 19C shows the results of the mNSS test. *p<0.05 for hUTC vs. vehicle (n=8/group). The statistic differences were based on the global test on all three tests.

FIG. 20. Graph showing neurological functional outcomes following MCAo in rats. FIG. 20 shows the neurological function outcomes for hUTC at doses of 3×10⁵ and 1×10⁶ administered 30 days after MCAo (see Example 24). FIG. 20A shows the results of the Adhesive Removal Test. FIG. 20B shows the results of the Foot-Fault Test and FIG. 20C shows the results of the mNSS test. *p<0.05 for (a) hUTC 1×10⁶ vs. vehicle (n=8/group). The statistic differences were based on the global test on all three tests.

FIG. 21. Graph showing body weight following MCAo in rats. FIG. 21 shows the body weight of rats post-MCAo (see Example 24). FIG. 21A shows the body weight of rats treated with hUTC with or without CsA (including controls) at 1 day after MCAo as a function of time. FIG. 21B shows the body weight of rats treated with hUTC at 15 days after MCAo as a function of time. FIG. 21C shows the body weight of rats treated with 3×10⁵ or 1×10⁶ of hUTC at 30 days after MCAo as a function of time.

FIG. 22. Graph showing lesion volume following MCAo in rats (see Example 24).

FIG. 23. Graph showing vessel density following MCAo in rats (see Example 24).

FIG. 24. Graph showing synaptophysin immunoreactivity following MCAo in rats (see Example 24).

FIG. 25. Graph showing the forelimb placing test results for intranasal delivery of hUTC after stroke in the rodent model for ischemic stroke (see Example 25).

FIG. 26. Graph showing the hindlimb placing test results for intranasal delivery of hUTC after stroke in the rodent model for ischemic stroke (see Example 25). In FIG. 26, **=different from vehicle-treated group by p<0.001.

FIG. 27. Graph showing the body swing test results for intranasal delivery of hUTC after stroke in the rodent model for ischemic stroke (see Example 25). In FIG. 27, ***=different from vehicle-treated group by <0.001.

FIG. 28. Graph showing the weight of rodents as a function of time (in days after stroke) in a study investigating intranasal delivery of hUTC after stroke in the rodent model for ischemic stroke (see Example 25).

FIGS. 29 and 30. Graphs showing the forelimb placing test results for a study investigating the intranasal delivery of hUTC after stroke in the rodent model for ischemic stroke (see Example 26). FIG. 29A shows overall results for the forelimb placing test. FIG. 29B shows the forelimb placing test results for HU, CSCV4 vs. HU, hUTC, 1 MM cells. FIG. 30 shows the forelimb placing test results for CSCV4 vs. hUTC, 1 MM cells. In FIG. 30, *=different from vehicle-treated group by p<0.05.

FIGS. 31 and 32. Graphs showing the hindlimb placing test results for a study investigating the intranasal delivery of hUTC after stroke in the rodent model for ischemic stroke (see Example 26). FIG. 31A shows overall results for the hindlimb placing test. FIG. 31B shows the forelimb placing test results for HU, CSCV4 vs. HU, hUTC, 1 MM cells. FIG. 32 shows the forelimb placing test results for CSCV4 vs. hUTC, 1 MM cells. In FIG. 32, *=different from vehicle-treated group by p<0.05.

FIGS. 33 and 34. Graphs showing the body swing test results for a study investigating the intranasal delivery of hUTC after stroke in the rodent model for ischemic stroke (see Example 26). FIG. 33A shows overall results for the body swing test. FIG. 33B shows the body swing test results for HU, CSCV4 vs. HU, hUTC, 1 MM cells. FIG. 34 shows the body swing test results for CSCV4 vs. hUTC, 1 MM cells. In FIGS. 33 and 34, **=different from vehicle-treated group by p<0.01 and ***=different from vehicle-treated group by <0.001.

FIGS. 35 and 36. Graphs showing weight of rodents as a function of time (in days after stroke) for a study investigating the intranasal delivery of hUTC after stroke in the rodent model for ischemic stroke (see Example 26). FIG. 35A shows overall results for weight of rodents as a function of time. FIG. 35B shows the weight of rodents as a function of time for HU, CSCV4 vs. HU, hUTC, 1 MM cells. FIG. 36 shows weight of rodents as a function of time for CSCV4 vs. hUTC, 1 MM cells.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

Stem cells are undifferentiated cells defined by the ability of a single cell both to self-renew, and to differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation, and to contribute substantially to most, if not all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as: (1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and (5) unipotent. Totipotent cells are able to give rise to all embryonic and extraembryonic cell types. Pluripotent cells are able to give rise to all embryonic cell types. Multipotent cells include those able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell-restricted oligopotent progenitors, and all cell types and elements (e.g., platelets) that are normal components of the blood). Cells that are oligopotent can give rise to a more restricted subset of cell lineages than multipotent stem cells; and cells that are unipotent are able to give rise to a single cell lineage (e.g., spermatogenic stem cells).

Stem cells are also categorized on the basis of the source from which they may be obtained. An adult stem cell is generally a multipotent undifferentiated cell found in tissue comprising multiple differentiated cell types. The adult stem cell can renew itself. Under normal circumstances, it can also differentiate to yield the specialized cell types of the tissue from which it originated, and possibly other tissue types. An embryonic stem cell is a pluripotent cell from the inner cell mass of a blastocyst-stage embryo. A fetal stem cell is one that originates from fetal tissues or membranes. A postpartum stem cell is a multipotent or pluripotent cell that originates substantially from extraembryonic tissue available after birth, namely, the placenta and the umbilical cord. These cells have been found to possess features characteristic of pluripotent stem cells, including rapid proliferation and the potential for differentiation into many cell lineages. Postpartum stem cells may be blood-derived (e.g., as are those obtained from umbilical cord blood) or non-blood-derived (e.g., as obtained from the non-blood tissues of the umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from the embryo (which in humans refers to the period from fertilization to about six weeks of development. Fetal tissue refers to tissue originating from the fetus, which in humans refers to the period from about six weeks of development to parturition. Extraembryonic tissue is tissue associated with, but not originating from, the embryo or fetus. Extraembryonic tissues include extraembryonic membranes (chorion, amnion, yolk sac and allantois), umbilical cord and placenta (which itself forms from the chorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell, such as a nerve cell or a muscle cell, for example. A differentiated cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term committed, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e. which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation.

In a broad sense, a progenitor cell is a cell that has the capacity to create progeny that are more differentiated than itself, and yet retains the capacity to replenish the pool of progenitors. By that definition, stem cells themselves are also progenitor cells, as are the more immediate precursors to terminally differentiated cells. When referring to the cells of the present invention, as described in greater detail below, this broad definition of progenitor cell may be used. In a narrower sense, a progenitor cell is often defined as a cell that is intermediate in the differentiation pathway, i.e., it arises from a stem cell and is intermediate in the production of a mature cell type or subset of cell types. This type of progenitor cell is generally not able to self-renew. Accordingly, if this type of cell is referred to herein, it will be referred to as a non-renewing progenitor cell or as an intermediate progenitor or precursor cell.

As used herein, the phrase differentiates into a neural lineage or phenotype refers to a cell that becomes partially or fully committed to a specific neural phenotype of the CNS or PNS, i.e., a neuron or a glial cell, the latter category including without limitation astrocytes, oligodendrocytes, Schwann cells and microglia.

The cells of the present invention are generally referred to as umbilical cord tissue-derived cells (or UTC or hUTCs). They also may sometimes be referred to as umbilicus-derived cells (UDCs). In addition, the cells may be described as being stem or progenitor cells, the latter term being used in the broad sense. The term derived is used to indicate that the cells have been obtained from their biological source and grown or otherwise manipulated in vitro (e.g., cultured in a Growth Medium to expand the population and/or to produce a cell line). The in vitro manipulations of umbilical stem cells and the unique features of the umbilicus-derived cells of the present invention are described in detail below.

Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition (“in culture” or “cultured”). A primary cell culture is a culture of cells, tissues, or organs taken directly from an organism(s) before the first subculture. Cells are expanded in culture when they are placed in a Growth Medium under conditions that facilitate cell growth and/or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is sometimes measured by the amount of time needed for the cells to double in number. This is referred to as doubling time.

A cell line is a population of cells formed by one or more subcultivations of a primary cell culture. Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore, the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but not limited to the seeding density, substrate, medium, growth conditions, and time between passaging.

A conditioned medium is a medium in which a specific cell or population of cells has been cultured, and then removed. When cells are cultured in a medium, they may secrete cellular factors that can provide trophic support to other cells. Such trophic factors include, but are not limited to hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. The medium containing the cellular factors is the conditioned medium.

Growth Medium generally refers to a medium sufficient for the culturing of UTC. In particular, one presently preferred medium for the culturing of the cells of the invention in comprises Dulbecco's Modified Eagle Media (also known as Dulbecco's Minimal Essential Media) (DMEM). Particularly preferred is DMEM-low glucose (also DMEM-LG herein) (Invitrogen, Carlsbad, Calif.). The DMEM-low glucose is preferably supplemented with 15% (v/v) fetal bovine serum (e.g., defined fetal bovine serum, Hyclone, Logan Utah), antibiotics and antimycotics (preferably, 50-100 Units/milliliter penicillin, 50-100 microgram/milliliter streptomycin, and 0-0.25 microgram/milliliter amphotericin B; Invitrogen, Carlsbad, Calif.), and 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.).

The term standard growth conditions refers to culturing of cells at 37° C., in a standard humidified atmosphere comprising 5% CO₂. While such conditions are useful for culturing, it is to be understood that such conditions are capable of being varied by the skilled artisan who will appreciate the options available in the art for culturing cells.

Generally, a trophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a cell, or stimulates increased activity of a cell. The interaction between cells via trophic factors may occur between cells of different types. Cell interaction by way of trophic factors is found in essentially all cell types, and is a particularly significant means of communication among neural cell types. Trophic factors also can function in an autocrine fashion, i.e., a cell may produce trophic factors that affect its own survival, growth, differentiation, proliferation and/or maturation.

When referring to cultured vertebrate cells, the term senescence (also replicative senescence or cellular senescence) refers to a property attributable to finite cell cultures; namely, their inability to grow beyond a finite number of population doublings (sometimes referred to as Hayflick's limit). Although cellular senescence was first described using fibroblast-like cells, most normal human cell types that can be grown successfully in culture undergo cellular senescence. The in vitro lifespan of different cell types varies, but the maximum lifespan is typically fewer than 100 population doublings (this is the number of doublings for all the cells in the culture to become senescent and thus render the culture unable to divide). Senescence does not depend on chronological time, but rather is measured by the number of cell divisions, or population doublings, the culture has undergone. Thus, cells made quiescent by removing essential growth factors are able to resume growth and division when the growth factors are re-introduced, and thereafter carry out the same number of doublings as equivalent cells grown continuously. Similarly, when cells are frozen in liquid nitrogen after various numbers of population doublings and then thawed and cultured, they undergo substantially the same number of doublings as cells maintained unfrozen in culture. Senescent cells are not dead or dying cells; they are actually resistant to programmed cell death (apoptosis), and have been maintained in their nondividing state for as long as three years. These cells are very much alive and metabolically active, but they do not divide. The nondividing state of senescent cells has not yet been found to be reversible by any biological, chemical, or viral agent.

The term neurodegenerative condition (or disorder) is an inclusive term encompassing acute and chronic conditions, disorders or diseases of the central or peripheral nervous system. A neurodegenerative condition may be age-related, or it may result from injury or trauma, or it may be related to a specific disease or disorder. Acute neurodegenerative conditions include, but are not limited to, conditions associated with neuronal cell death or compromise including cerebrovascular insufficiency, e.g. due to stroke, focal or diffuse brain trauma, diffuse brain damage, spinal cord injury or peripheral nerve trauma, e.g., resulting from physical or chemical burns, deep cuts or limb severance. Examples of acute neurodegenerative disorders are: cerebral ischemia or infarction including embolic occlusion and thrombotic occlusion, reperfusion following acute ischemia, perinatal hypoxic-ischemic injury, cardiac arrest, as well as intracranial hemorrhage of any type (such as epidural, subdural, subarachnoid and intracerebral), and intracranial and intravertebral lesions (such as contusion, penetration, shear, compression and laceration), as well as whiplash and shaken infant syndrome. Chronic neurodegenerative conditions include, but are not limited to, Alzheimer's disease, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Drager syndrome), chronic epileptic conditions associated with neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxias, cortical basal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies (including multiple system atrophy), primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Gilles De La Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sach's disease, Sandhoff disease, familial spastic disease, Wohlfart-Kugelberg-Welander disease, spastic paraparesis, progressive multifocal leukoencephalopathy, familial dysautonomia (Riley-Day syndrome), and prion diseases (including, but not limited to Creutzfeldt-Jakob, Gerstmann-Sträussler-Scheinker disease, Kuru and fatal familial insomnia), demyelination diseases and disorders including multiple sclerosis and hereditary diseases such as leukodystrophies.

Other neurodegenerative conditions include tumors and other neoplastic conditions affecting the CNS and PNS. Though the underlying disease is considered proliferative (rather than neurodegenerative), surrounding tissues may be compromised. Furthermore, cell therapy may be utilized to deliver apoptotic or other antineoplastic molecules to the tumor site, e.g., via delivery of genetically modified cells producing such agents.

Other neurodegenerative conditions include various neuropathies, such as multifocal neuropathies, sensory neuropathies, motor neuropathies, sensory-motor neuropathies, infection-related neuropathies, autonomic neuropathies, sensory-autonomic neuropathies, demyelinating neuropathies (including, but not limited to, Guillain-Barre syndrome and chronic inflammatory demyelinating polyradiculoneuropathy), other inflammatory and immune neuropathies, neuropathies induced by drugs, neuropathies induced by pharmacological treatments, neuropathies induced by toxins, traumatic neuropathies (including, but not limited to, compression, crush, laceration and segmentation neuropathies), metabolic neuropathies, endocrine and paraneoplastic neuropathies, among others.

The term stroke refers to any condition arising from a disruption, decrease, or stoppage of blood or oxygen flow to any part of the brain. “Ischemic stroke” refers to a stroke resulting from any disruption, decrease or stoppage in the blood supply to any part of the brain caused by any constriction or obstruction of the vasculature. The obstruction of vasculature may be either temporal or permanent. “Hemorrhagic stroke” refers to a stroke resulting from any rupture in any of the vasculature of the brain. Examples of acute neurodegenerative disorders that include stroke or involve etiology or symptoms such as those observed with stroke are listed above, and include: cerebral ischemia or infarction including embolic occlusion and thrombotic occlusion, reperfusion following acute ischemia, perinatal hypoxic-ischemic injury, cardiac arrest, as well as intracranial hemorrhage of any type (such as epidural, subdural, subarachnoid and intracerebral), and intracranial and intravertebral lesions (such as contusion, penetration, shear, compression and laceration), as well as whiplash and shaken infant syndrome.

Other neurodegenerative conditions include dementias, regardless of underlying etiology, including age-related dementia and other dementias and conditions with memory loss including dementia associated with Alzheimer's disease, vascular dementia, diffuse white matter disease (Binswanger's disease), dementia of endocrine or metabolic origin, dementia of head trauma and diffuse brain damage, dementia pugilistica and frontal lobe dementia.

The term treating (or treatment of) a neurodegenerative condition refers to ameliorating the effects of, or delaying, halting or reversing the progress of, or delaying or preventing the onset of, a neurodegenerative condition as defined herein.

The term effective amount refers to a concentration or amount of a reagent or pharmaceutical composition, such as a growth factor, differentiation agent, trophic factor, cell population or other agent, that is effective for producing an intended result, including cell growth and/or differentiation in vitro or in vivo, or treatment of a neurodegenerative condition as described herein. Such results include, but are not limited to, the regeneration, repair, or improvement of neural tissue, and/or the improvement of behavior and neurological function in stroke patients. Such effective activity may be achieved, for example, by administering the cells and/or compositions of the present invention to a stroke patient. With respect to growth factors, an effective amount may range from about 1 nanogram/milliliter to about 1 microgram/milliliter. With respect to cells as administered to a patient in vivo, an effective amount may range from as few as several hundred or fewer to as many as several million or more. In specific embodiments, an effective amount may range from 10³-10¹¹, more specifically at least about 10⁴ cells. It will be appreciated that the number of cells to be administered will vary depending on the specifics of the disorder to be treated, including but not limited to size or total volume/surface area to be treated, as well as proximity of the site of administration to the location of the region to be treated, among other factors familiar to the skilled artisan.

The terms effective period (or time) and effective conditions refer to a period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods), necessary or preferred for an agent or pharmaceutical composition to achieve its intended result.

The terms effective period (or time) and effective conditions refer to a period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods), necessary or preferred for an agent or pharmaceutical composition to achieve its intended result.

The term pharmaceutically acceptable carrier (or medium), which may be used interchangeably with the term biologically compatible carrier or medium, refers to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio. As described in greater detail herein, pharmaceutically acceptable carriers suitable for use in the present invention include liquids, semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds and matrices, tubes sheets and other such materials as known in the art and described in greater detail herein). These semi-solid and solid materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable). A biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathways.

Several terms are used herein with respect to cell or tissue transplantation. The terms autologous transfer, autologous transplantation, autograft and the like refer to transplantation wherein the transplant donor is also the transplant recipient. The terms allogeneic transfer, allogeneic transplantation, allograft and the like refer to transplantation wherein the transplant donor is of the same species as the transplant recipient, but is not the same individual. A cell transplant in which the donor's cells and have been histocompatibility matched with a recipient is sometimes referred to as a syngeneic transfer. The terms xenogeneic transfer, xenogeneic transplantation, xenograft and the like refer to transplantation wherein the transplant donor is of a different species than the transplant recipient.

Description

Neurodegenerative conditions, which encompass acute, chronic and progressive disorders and diseases having widely divergent causes, have as a common feature the dysfunction or loss of a specific or vulnerable group of neural cells. This commonality enables development of similar therapeutic approaches for the repair and regeneration of vulnerable or damaged neural tissue, one of which is cell-based therapy. In its various embodiments described herein, the present invention features methods and pharmaceutical compositions for neural repair and regeneration that utilize progenitor cells and cell populations derived from umbilical cord tissue. The invention is applicable to any neurodegenerative condition, but is expected to be particularly suitable for the treatment of strokes (such as e.g. ischemic and/or hemorrhagic strokes).

As summarized above, the invention, in one of its aspects is generally directed to isolated umbilical cord tissue-derived cells (UTCs), which have been rendered substantially free of blood. The UTCs are capable of self-renewal and expansion in culture and have the potential to differentiate into cells of neural phenotypes. Certain embodiments feature populations comprising such cells, pharmaceutical compositions comprising the cells or components or products thereof, and methods of using the pharmaceutical compositions for treatment of patients with acute or chronic neurodegenerative conditions (such as e.g. stroke). The umbilical cord tissue-derived cells have been characterized by their growth properties in culture, by their cell surface markers, by their gene expression, by their ability to produce certain biochemical trophic factors, and by their immunological properties.

Preparation of Umbilicus and Placenta Derived Tissue Cells

According to the methods described herein, a mammalian placenta and umbilical cord are recovered upon or shortly after termination of either a full-term or pre-term pregnancy, for example, after expulsion after birth. The tissue may be transported from the birth site to a laboratory in a sterile container such as a flask, beaker, culture dish, or bag. The container may have a solution or medium, including but not limited to a salt solution, such as, for example, Dulbecco's Modified Eagle's Medium (DMEM) (also known as Dulbecco's Minimal Essential Medium) or phosphate buffered saline (PBS), or any solution used for transportation of organs used for transplantation, such as University of Wisconsin solution or perfluorochemical solution. One or more antibiotic and/or antimycotic agents, such as but not limited to penicillin, streptomycin, amphotericin B, gentamicin, and nystatin, may be added to the medium or buffer. The umbilical and placenta tissue may be rinsed with an anticoagulant solution such as heparin-containing solution. It is preferable to keep the tissue at about 4-10° C. prior to extraction of the cells. It is even more preferable that the tissue not be frozen prior to extraction of the umbilical or placenta-derived cells.

Isolation of cells preferably occurs in an aseptic environment. The umbilical cord may be separated from the placenta by means known in the art. Alternatively, the umbilical cord and placenta are used without separation. Blood and debris are preferably removed from the tissue prior to isolation of cells. For example, the tissue may be washed with buffer solution, such as but not limited to phosphate buffered saline. The wash buffer also may comprise one or more antimycotic and/or antibiotic agents, such as but not limited to penicillin, streptomycin, amphotericin B, gentamicin, and nystatin.

Tissue comprising a whole umbilical cord, whole placenta or a fragment or section thereof is disaggregated by mechanical force (mincing or shear forces). In a presently preferred embodiment, the isolation procedure also utilizes an enzymatic digestion process. Many enzymes are known in the art to be useful for the isolation of individual cells from complex tissue matrices to facilitate growth in culture. Ranging from weakly digestive (e.g. deoxyribonucleases and the neutral protease, dispase) to strongly digestive (e.g. papain and trypsin), such enzymes are available commercially. A nonexhaustive list of enzymes compatible herewith includes mucolytic enzyme activities, metalloproteases, neutral proteases, serine proteases (such as trypsin, chymotrypsin, or elastase), and deoxyribonucleases. Presently preferred are enzyme activities selected from metalloproteases, neutral proteases and mucolytic activities. For example, collagenases are known to be useful for isolating various cells from tissues. Deoxyribonucleases can digest single-stranded DNA and can minimize cell-clumping during isolation. Preferred methods involve enzymatic treatment with for example collagenase and dispase, or collagenase, dispase, and hyaluronidase, and such methods are provided wherein in certain preferred embodiments, a mixture of collagenase and the neutral protease dispase are used in the dissociating step. More preferred are those methods which employ digestion in the presence of at least one collagenase from Clostridium histolyticum, and either of the protease activities, dispase and thermolysin. Still more preferred are methods employing digestion with both collagenase and dispase enzyme activities. Also preferred are methods which include digestion with a hyaluronidase activity in addition to collagenase and dispase activities. The skilled artisan will appreciate that many such enzyme treatments are known in the art for isolating cells from various tissue sources. For example, the LIBERASE™ Blendzyme (Roche) series of enzyme combinations are suitable for use in the instant methods. Other sources of enzymes are known, and the skilled artisan may also obtain such enzymes directly from their natural sources. The skilled artisan is also well-equipped to assess new, or additional enzymes or enzyme combinations for their utility in isolating the cells of the invention. Preferred enzyme treatments are 0.5, 1, 1.5, or 2 hours long or longer. In other preferred embodiments, the tissue is incubated at 37° C. during the enzyme treatment of the dissociation step.

In some embodiments of the invention, the tissue is separated into sections comprising various aspects of the tissue, such as neonatal, neonatal/maternal, and maternal aspects of the placenta, for instance. The separated sections then are dissociated by mechanical and/or enzymatic dissociation according to the methods described herein. Cells of neonatal or maternal lineage may be identified by any means known in the art, for example, by karyotype analysis or in situ hybridization for a Y chromosome.

Isolated cells or tissue from which hUTCs grow out may be used to initiate, or seed, cell cultures. Isolated cells are transferred to sterile tissue culture vessels either uncoated or coated with extracellular matrix or ligands such as laminin, collagen (native, denatured or crosslinked), gelatin, fibronectin, and other extracellular matrix proteins. hUTCs are cultured in any culture medium capable of sustaining growth of the cells such as, but not limited to, DMEM (high or low glucose), advanced DMEM, DMEM/MCDB 201, Eagle's basal medium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM), DMEM/F12, RPMI 1640, and CELL-GRO-FREE™. The culture medium may be supplemented with one or more components including, for example, fetal bovine serum (FBS), preferably about 2-15% (v/v); equine serum (ES); human serum (HS); beta-mercaptoethanol (BME or 2-ME), preferably about 0.001% (v/v); one or more growth factors, for example, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), leukocyte inhibitory factor (LIF) and erythropoietin; amino acids, including L-valine; and one or more antibiotic and/or antimycotic agents to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination. The culture medium preferably comprises Growth Medium (DMEM-low glucose, serum, BME, and an antibiotic agent) as defined in the Examples below.

The cells are seeded in culture vessels at a density to allow cell growth. In a preferred embodiment, the cells are cultured at about 0 to about 5 percent by volume CO₂ in air. In some preferred embodiments, the cells are cultured at about 2 to about 25 percent O₂ in air, preferably about 5 to about 20 percent O₂ in air. The cells preferably are cultured at about 25 to about 40° C. and more preferably are cultured at 37° C. The cells are preferably cultured in an incubator. The medium in the culture vessel can be static or agitated, for example, using a bioreactor. The cells preferably are grown under low oxidative stress (e.g., with addition of glutathione, Vitamin C, Catalase, Vitamin E, N-Acetylcysteine). “Low oxidative stress”, as used herein, refers to conditions of no or minimal free radical damage to the cultured cells.

Methods for the selection of the most appropriate culture medium, medium preparation, and cell culture techniques are well known in the art and are described in a variety of sources, including Doyle et al., (eds.), 1995, CELL & TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley & Sons, Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS, Butterworth-Heinemann, Boston, which are incorporated herein by reference.

After culturing the isolated cells or tissue fragments for a sufficient period of time, UTCs will have grown out, either as a result of migration from the tissue or cell division, or both. In some embodiments of the invention, UTCs are passaged, or removed to a separate culture vessel containing fresh medium of the same or a different type as that used initially, where the population of cells can be mitotically expanded. The cells of the invention may be used at any point between passage 0 and senescence. The cells preferably are passaged between about 3 and about 25 times, more preferably are passaged about 4 to about 12 times, and preferably are passaged 10 or 11 times. Cloning and/or subcloning may be performed to confirm that a clonal population of cells has been isolated.

In some aspects of the invention, the different cell types present in umbilical cord or placental tissue are fractionated into subpopulations from which the cells can be isolated. This may be accomplished using standard techniques for cell separation including, but not limited to, enzymatic treatment to dissociate the tissue into its component cells, followed by cloning and selection of specific cell types, for example but not limited to selection based on morphological and/or biochemical markers; selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); separation based upon differential cell agglutinability in the mixed population as, for example, with soybean agglutinin; freeze-thaw procedures; differential adherence properties of the cells in the mixed population; filtration; conventional and zonal centrifugation; centrifugal elutriation (counter-streaming centrifugation); unit gravity separation; countercurrent distribution; electrophoresis; and fluorescence activated cell sorting (FACS). For a review of clonal selection and cell separation techniques, see Freshney, 1994, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, 3rd Ed., Wiley-Liss, Inc., New York, which is incorporated herein by reference.

The culture medium is changed as necessary, for example, by carefully aspirating the medium from the dish, for example, with a pipette, and replenishing with fresh medium. Incubation is continued until a sufficient number or density of cells accumulates in the dish. The original explanted tissue sections may be removed and the remaining cells trypsinized using standard techniques or using a cell scraper or non-enzymatic means of cell removal such as solutions contain chelator or on cell culture dishes coated with polymers allowing easy detachment. After trypsinization, the cells are collected, removed to fresh medium and incubated as above. In some embodiments, the medium is changed at least once at approximately 24 hours post-trypsinization to remove any floating cells. The cells remaining in culture are considered to be UTCs.

UTCs may be cryopreserved. Accordingly, in a preferred embodiment described in greater detail below, UTCs for autologous transfer (for either the mother or child) may be derived from appropriate postpartum tissues following the birth of a child, then cryopreserved so as to be available in the event they are later needed for transplantation.

Characteristics of Umbilicus- and Placenta-Derived Cells

Umbilicus- and placenta-derived cells may be characterized, for example, by growth characteristics (e.g., population doubling capability, doubling time, passages to senescence), karyotype analysis (e.g., normal karyotype; maternal or neonatal lineage), flow cytometry (e.g., FACS analysis), immunohistochemistry and/or immunocytochemistry (e.g., for detection of epitopes), gene expression profiling (e.g., gene chip arrays; polymerase chain reaction (for example, reverse transcriptase PCR, real time PCR, and conventional PCR)), protein arrays, protein secretion (e.g., by plasma clotting assay or analysis of PDC-conditioned medium, for example, by Enzyme Linked ImmunoSorbent Assay (ELISA)), mixed lymphocyte reaction (e.g., as measure of stimulation of PBMCs), and/or other methods known in the art.

Examples of cells derived from placental tissue were deposited with the American Type Culture Collection (ATCC, Manassas, Va.) and assigned ATCC Accession Numbers as follows: (1) strain designation PLA 071003 (P8) was deposited Jun. 15, 2004 and assigned Accession No. PTA-6074; (2) strain designation PLA 071003 (P11) was deposited Jun. 15, 2004 and assigned Accession No. PTA-6075; and (3) strain designation PLA 071003 (P16) was deposited Jun. 16, 2004 and assigned Accession No. PTA-6079. Examples of cells derived from umbilicus tissue were deposited with the American Type Culture Collection on Jun. 10, 2004, and assigned ATCC Accession Numbers as follows: (1) strain designation UMB 022803 (P7) was assigned Accession No. PTA-6067; and (2) strain designation UMB 022803 (P17) was assigned Accession No. PTA-6068.

In various embodiments, the cells possess one or more of the following growth features (1) they require L-valine for growth in culture; (2) they are capable of growth in atmospheres containing oxygen from about 5% to at least about 20% (3) they have the potential for at least about 40 doublings in culture before reaching senescence; and (4) they attach and expand on a coated or uncoated tissue culture vessel, wherein the coated tissue culture vessel comprises a coating of gelatin, laminin, collagen, polyornithine, vitronectin or fibronectin.

In certain embodiments the cells possess a normal karyotype, which is maintained as the cells are passaged. Karyotyping is particularly useful for identifying and distinguishing neonatal from maternal cells derived from placenta. Methods for karyotyping are available and known to those of skill in the art.

In other embodiments, the cells may be characterized by production of certain proteins, including (1) production of at least one of tissue factor, vimentin, and alpha-smooth muscle actin; and (2) production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C cell surface markers, as detected by flow cytometry. In other embodiments, the UTCs may be characterized by lack of production of at least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ cell surface markers, as detected by flow cytometry. Particularly preferred are cells that produce at least two of tissue factor, vimentin, and alpha-smooth muscle actin. More preferred are those cells producing all three of the proteins tissue factor, vimentin, and alpha-smooth muscle actin.

In other embodiments, the cells may be characterized by gene expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, is increased for a gene encoding at least one of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; tumor necrosis factor and alpha-induced protein 3.

In yet other embodiments, the cells may be characterized by gene expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell, is reduced for a gene encoding at least one of: short stature homeobox 2; heat shock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1); elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeo box 2 (growth arrest-specific homeo box); sine oculis homeobox homolog 1 (Drosophila); crystallin, alpha B; disheveled associated activator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogen binding protein); src homology three (SH3) and cysteine rich domain; cholesterol 25-hydroxylase; runt-related transcription factor 3; interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein 5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2; neuroblastoma, suppression of tumorigenicity 1; insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNA F1112280 fis, clone MAMMA1001744; cytokine receptor-like factor 1; potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4; integrin, beta 7; transcriptional co-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin); EGF-containing fibulin-like extracellular matrix protein 1; early growth response 3; distal-less homeo box 5; hypothetical protein F1120373; aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan; transcriptional co-activator with PDZ-binding motif (TAZ); fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE binding protein 1; and cytochrome c oxidase subunit VIIa polypeptide 1 (muscle).

In other embodiments, the cells may be characterized by secretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1. In alternative embodiments, the cells may be characterized by lack of secretion of at least one of TGF-beta2, ANG2, PDGFbb, MIP1b, 1309, MDC, and VEGF, as detected by ELISA.

In preferred embodiments, the cell comprises two or more of the above-listed growth, protein/surface marker production, gene expression or substance-secretion characteristics. More preferred are those cells comprising, three, four, or five or more of the characteristics. Still more preferred are cells comprising six, seven, or eight or more of the characteristics. Still more preferred presently are those cells comprising all of above characteristics.

In preferred embodiments, the cells do not express telomerase (hTert). Accordingly, one embodiment of the invention is umbilical-derived cells that do not express telomerase (hTert) and that have one or more of the characteristics disclosed herein.

In one embodiment of the invention, the cells are umbilical cord tissue-derived cells which are isolated from human umbilical cord tissue substantially free of blood, are capable of self-renewal and expansion into culture, have the potential to differentiate into cells of other phenotypes, can undergo at least 40 doublings, and have the following characteristics: (a) express each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C; (b) do not express any of CD31, CD34, CD45, CD80, CD86, CD 117, CD141, CD178, B7-H2, HLA-G, or HLA-DR,DP,DQ; and (c) increased expression of interleukin-8; reticulon 1; and chemokine receptor ligand (C-X-C motif) ligand 3, relative to that of a human cell which is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell. In one embodiment, these umbilical cord derived cells also have one of more of the following characteristics: (a) secretion of each of the factor MCP-1, MIP1beta, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, RANTES, and TIMP1; and (b) no secretion of any of the factors SDF-1alpha TGF-beta2, ANG2, PDGFbb, MIP1a and VEGF. In another embodiment, these umbilical cord tissue-derived cells do not express hTERT or telomerase.

In another embodiment, the cells are umbilical cord tissue-derived cells which are isolated from human umbilical cord tissue substantially free of blood, are capable of self-renewal and expansion into culture, have the potential to differentiate into cells of other phenotypes, do not express CD117 and express telomerase or hTert. In yet another embodiment, the cells further do not express CD45. In an alternate embodiment, the cells further do not express any of CD31, CD34, CD80, CD86, CD141, CD178, B7-H2, HLA-G, or HLA-DR,DP,DQ. In another alternate embodiment, the cells further express each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C. In yet another embodiment of the invention, the cells further can undergo at least 40 doublings. In yet another embodiment, the cells further show increased expression of interleukin-8; reticulon 1; and chemokine receptor ligand (C-X-C motif) ligand 3, relative to that of a human cell which is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell. In yet another embodiment, the cells further have each of the following characteristics: (a) secretion of each of the factor MCP-1, MIP1beta, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, RANTES, and TIMP1 and (b) no secretion of any of the factors SDF-1alpha TGF-beta2, ANG2, PDGFbb, MIP1a and VEGF.

Among cells that are presently preferred for use with the invention in several of its aspects are umbilical cord tissue-derived cells having the characteristics described above and more particularly those wherein the cells have normal karyotypes and maintain normal karyotypes with passaging, and further wherein the cells express each of the markers CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-A,B,C, wherein the cells produce the immunologically-detectable proteins which correspond to the listed markers. Still more preferred are those cells which in addition to the foregoing do not produce proteins corresponding to any of the markers CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.

Certain cells having the potential to differentiate along lines leading to various phenotypes are unstable and thus can spontaneously differentiate. Presently preferred for use with the invention are cells that do not spontaneously differentiate, for example along neural lines. Preferred cells, when grown in Growth Medium, are substantially stable with respect to the cell markers produced on their surface, and with respect to the expression pattern of various genes, for example as determined using an Affymetrix GENECHIP®. The cells remain substantially constant, for example in their surface marker characteristics over passaging, through multiple population doublings.

However, one feature of the cells is that they may be deliberately induced to differentiate into neural lineage phenotypes by subjecting them to differentiation-inducing cell culture conditions. This may be accomplished by one or more methods known in the art. For instance, as exemplified herein, umbilical cord tissue-derived cells may be plated on flasks coated with laminin in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement, Invitrogen), L-glutamine and Penicillin/Streptomycin, the combination of which is referred to herein as Neural Progenitor Expansion (NPE) medium. NPE media may be further supplemented with bFGF and/or EGF. Alternatively, the UTCs may be induced to differentiate in vitro by (1) co-culturing the UTCs with neural progenitor cells, or (2) growing the UTCs in neural progenitor cell-conditioned medium.

Differentiation of the UTCs may be demonstrated by a bipolar cell morphology with extended processes. The induced cell populations may stain positive for the presence of nestin. Differentiated UTCs may be assessed by detection of nestin, TuJ1 (BIII tubulin), GFAP, tyrosine hydroxylase, GABA, O4 and/or MBP. In some embodiments, UTCs have exhibited the ability to form three-dimensional bodies characteristic of neuronal stem cell formation of neurospheres.

UTC Populations, Modifications, Components and Products

Another aspect of the invention features populations of the UTCs described above. In some embodiments, the cell population is heterogeneous. A heterogeneous cell population of the invention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% UTCs of the invention. The heterogeneous cell populations of the invention may further comprise stem cells or other progenitor cells, such as neural progenitor cells, or it may further comprise fully differentiated neural cells. In some embodiments, the population is substantially homogeneous, i.e., comprises substantially only UTCs (preferably at least about 96%, 97%, 98%, 99% or more UTCs). The homogeneous cell population of the invention comprises umbilicus-derived cells. Homogeneous populations of umbilicus-derived cells are preferably free of cells of maternal lineage. Homogeneity of a cell population may be achieved by any method known in the art, for example, by cell sorting (e.g., flow cytometry) or by clonal expansion in accordance with known methods. Thus, preferred homogeneous UTC populations may comprise a clonal cell line of umbilical cord tissue-derived cells. Such populations are particularly useful when a cell clone with highly desirable functionality has been isolated.

Also provided herein are populations of cells incubated in the presence of one or more factors, or under conditions, that stimulate stem cell differentiation along a neurogenic pathway. Such factors are known in the art and the skilled artisan will appreciate that determination of suitable conditions for differentiation can be accomplished with routine experimentation. Optimization of such conditions can be accomplished by statistical experimental design and analysis, for example response surface methodology allows simultaneous optimization of multiple variables, for example in a biological culture. Presently preferred factors include, but are not limited to factors, such as growth or trophic factors, demethylating agents, co-culture with neural lineage cells or culture in neural lineage cell-conditioned medium, as well other conditions known in the art to stimulate stem cell differentiation along a neurogenic pathway or lineage (see, e.g., Lang, K. J. D. et al., 2004, J. Neurosci. Res. 76: 184-192; Johe, K. K. et al., 1996, Genes Devel. 10: 3129-3140; Gottleib, D., 2002, Ann. Rev. Neurosci. 25: 381-407).

UTCs may also be genetically modified to produce neurotherapeutically useful gene products, or to produce antineoplastic agents for treatment of tumors, for example. Genetic modification may be accomplished using any of a variety of vectors including, but not limited to, integrating viral vectors, e.g., retrovirus vector or adeno-associated viral vectors; non-integrating replicating vectors, e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors; or replication-defective viral vectors. Other methods of introducing DNA into cells include the use of liposomes, electroporation, a particle gun, or by direct DNA injection.

Hosts cells are preferably transformed or transfected with DNA controlled by or in operative association with, one or more appropriate expression control elements such as promoter or enhancer sequences, transcription terminators, polyadenylation sites, among others, and a selectable marker. Any promoter may be used to drive the expression of the inserted gene. For example, viral promoters include, but are not limited to, the CMV promoter/enhancer, SV 40, papillomavirus, Epstein-Barr virus or elastin gene promoter. In some embodiments, the control elements used to control expression of the gene of interest can allow for the regulated expression of the gene so that the product is synthesized only when needed in vivo. If transient expression is desired, constitutive promoters are preferably used in a non-integrating and/or replication-defective vector. Alternatively, inducible promoters could be used to drive the expression of the inserted gene when necessary. Inducible promoters include, but are not limited to, those associated with metallothionein and heat shock proteins.

Following the introduction of the foreign DNA, engineered cells may be allowed to grow in enriched media and then switched to selective media. The selectable marker in the foreign DNA confers resistance to the selection and allows cells to stably integrate the foreign DNA as, for example, on a plasmid, into their chromosomes and grow to form foci which, in turn, can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the gene product.

The cells of the invention may be genetically engineered to “knock out” or “knock down” expression of factors that promote inflammation or rejection at the implant site. Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below. “Negative modulation,” as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment. The expression of a gene native to a neuron or glial cell can be reduced or knocked out using a number of techniques including, for example, inhibition of expression by inactivating the gene using the homologous recombination technique. Typically, an exon encoding an important region of the protein (or an exon 5′ to that region) is interrupted by a positive selectable marker, e.g., neo, preventing the production of normal mRNA from the target gene and resulting in inactivation of the gene. A gene may also be inactivated by creating a deletion in part of a gene, or by deleting the entire gene. By using a construct with two regions of homology to the target gene that are far apart in the genome, the sequences intervening the two regions can be deleted (Mombaerts et al., 1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084-3087). Antisense, DNAzymes, ribozymes, small interfering RNA (siRNA) and other such molecules that inhibit expression of the target gene can also be used to reduce the level of target gene activity. For example, antisense RNA molecules that inhibit the expression of major histocompatibility gene complexes (HLA) have been shown to be most versatile with respect to immune responses. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. These techniques are described in detail by L. G. Davis et al. (eds), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd ed., Appleton & Lange, Norwalk, Conn.

In other aspects, the invention provides cell lysates and cell soluble fractions prepared from UTCs, or heterogeneous or homogeneous cell populations comprising UTCs, as well as UTCs or populations thereof that have been genetically modified or that have been stimulated to differentiate along a neurogenic pathway. Such lysates and fractions thereof have many utilities. Use of the UTC lysate soluble fraction (i.e., substantially free of membranes) in vivo, for example, allows the beneficial intracellular milieu to be used allogeneically in a patient without introducing an appreciable amount of the cell surface proteins most likely to trigger rejection, or other adverse immunological responses. Methods of lysing cells are well-known in the art and include various means of mechanical disruption, enzymatic disruption, thermal, or chemical disruption, or combinations thereof. Such cell lysates may be prepared from cells directly in their Growth Medium and thus containing secreted growth factors and the like, or may be prepared from cells washed free of medium in, for example, PBS or other solution. Washed cells may be resuspended at concentrations greater than the original population density if preferred.

In one embodiment, whole cell lysates are prepared, e.g., by disrupting cells without subsequent separation of cell fractions. In another embodiment, a cell membrane fraction is separated from a soluble fraction of the cells by routine methods known in the art, e.g., centrifugation, filtration, or similar methods.

Cell lysates or cell soluble fractions prepared from populations of umbilical cord tissue-derived cells may be used as is, further concentrated, by for example, ultrafiltration or lyophilization, or even dried, partially purified, combined with pharmaceutically-acceptable carriers or diluents as are known in the art, or combined with other compounds such as biologicals, for example pharmaceutically useful protein compositions. Cell lysates or fractions thereof may be used in vitro or in vivo, alone or for example, with autologous or syngeneic live cells. The lysates, if introduced in vivo, may be introduced locally at a site of treatment, or remotely to provide, for example needed cellular growth factors to a patient.

In a further embodiment, UTCs can be cultured in vitro to produce biological products in high yield. For example, such cells, which either naturally produce a particular biological product of interest (e.g., a trophic factor), or have been genetically engineered to produce a biological product, can be clonally expanded using the culture techniques described herein. Alternatively, cells may be expanded in a medium that induces differentiation to a neural lineage. In either case, biological products produced by the cell and secreted into the medium can be readily isolated from the conditioned medium using standard separation techniques, e.g., such as differential protein precipitation, ion-exchange chromatography, gel filtration chromatography, electrophoresis, and HPLC, to name a few. A “bioreactor” may be used to take advantage of the flow method for feeding, for example, a three-dimensional culture in vitro. Essentially, as fresh media is passed through the three-dimensional culture, the biological product is washed out of the culture and may then be isolated from the outflow, as above.

Alternatively, a biological product of interest may remain within the cell and, thus, its collection may require that the cells be lysed, as described above. The biological product may then be purified using any one or more of the above-listed techniques.

In other embodiments, the invention provides conditioned medium from cultured UTCs for use in vitro and in vivo as described below. Use of the UTC conditioned medium allows the beneficial trophic factors secreted by the UTCs to be used allogeneically in a patient without introducing intact cells that could trigger rejection, or other adverse immunological responses. Conditioned medium is prepared by culturing cells in a culture medium, then removing the cells from the medium.

Conditioned medium prepared from populations of umbilical cord tissue-derived cells may be used as is, further concentrated, by for example, ultrafiltration or lyophilization, or even dried, partially purified, combined with pharmaceutically-acceptable carriers or diluents as are known in the art, or combined with other compounds such as biologicals, for example pharmaceutically useful protein compositions. Conditioned medium may be used in vitro or in vivo, alone or for example, with autologous or syngeneic live cells. The conditioned medium, if introduced in vivo, may be introduced locally at a site of treatment, or remotely to provide, for example needed cellular growth or trophic factors to a patient.

In another embodiment, an extracellular matrix (ECM) produced by culturing UTCs on liquid, solid or semi-solid substrates is prepared, collected and utilized as an alternative to implanting live cells into a subject in need of tissue repair or replacement. UTCs are cultured in vitro, on a three dimensional framework as described elsewhere herein, under conditions such that a desired amount of ECM is secreted onto the framework. The comprising the new tissue are removed, and the ECM processed for further use, for example, as an injectable preparation. To accomplish this, cells on the framework are killed and any cellular debris removed from the framework. This process may be carried out in a number of different ways. For example, the living tissue can be flash-frozen in liquid nitrogen without a cryopreservative, or the tissue can be immersed in sterile distilled water so that the cells burst in response to osmotic pressure.

Once the cells have been killed, the cellular membranes may be disrupted and cellular debris removed by treatment with a mild detergent rinse, such as EDTA, CHAPS or a zwitterionic detergent. Alternatively, the tissue can be enzymatically digested and/or extracted with reagents that break down cellular membranes and allow removal of cell contents. Examples of such enzymes include, but are not limited to, hyaluronidase, dispase, proteases, and nucleases. Examples of detergents include non-ionic detergents such as, for example, alkylaryl polyether alcohol (TRITON X-100), octylphenoxy polyethoxy-ethanol (Rohm and Haas Philadelphia, Pa.), BRIJ-35, a polyethoxyethanol lauryl ether (Atlas Chemical Co., San Diego, Calif.), polysorbate 20 (TWEEN 20), a polyethoxyethanol sorbitan monolaureate (Rohm and Haas), polyethylene lauryl ether (Rohm and Haas); and ionic detergents such as, for example, sodium dodecyl sulfate (SDS), sulfated higher aliphatic alcohols, sulfonated alkanes and sulfonated alkylarenes containing 7 to 22 carbon atoms in a branched or unbranched chain.

The collection of the ECM can be accomplished in a variety of ways, depending, for example, on whether the new tissue has been formed on a three-dimensional framework that is biodegradable or non-biodegradable. For example, if the framework is non-biodegradable, the ECM can be removed by subjecting the framework to sonication, high pressure water jets, mechanical scraping, or mild treatment with detergents or enzymes, or any combination of the above.

If the framework is biodegradable, the ECM can be collected, for example, by allowing the framework to degrade or dissolve in solution. Alternatively, if the biodegradable framework is composed of a material that can itself be injected along with the ECM, the framework and the ECM can be processed in toto for subsequent injection. Alternatively, the ECM can be removed from the biodegradable framework by any of the methods described above for collection of ECM from a non-biodegradable framework. All collection processes are preferably designed so as not to denature the ECM. A non-biodegradable framework can be implanted with the cells.

After it has been collected, the ECM may be processed further. For example, the ECM can be homogenized to fine particles using techniques well known in the art such as by sonication, so that it can pass through a surgical needle. The components of the ECM can be crosslinked, if desired, by gamma irradiation. Preferably, the ECM can be irradiated between 0.25 to 2 mega rads to sterilize and crosslink the ECM. Chemical crosslinking using agents that are toxic, such as glutaraldehyde, is possible but not generally preferred.

The amounts and/or ratios of proteins, such as the various types of collagen present in the ECM, may be adjusted by mixing the ECM produced by the cells of the invention with ECM of one or more other cell types. In addition, biologically active substances such as proteins, growth factors and/or drugs, can be incorporated into the ECM. Exemplary biologically active substances include tissue growth factors, such as TGF-beta, and the like, which promote healing and tissue repair at the site of the injection, and BDNF, which promotes neural function and protection. Such additional agents may be utilized in any of the embodiments described herein above, e.g., with whole cell lysates, soluble cell fractions, or further purified components and products produced by the UTCs.

Pharmaceutical Compositions Comprising UTCs, UTC Components or Products

In another aspect, the invention provides pharmaceutical compositions that utilize the UTCs, UTC populations, components and products of UTCs in various methods for treatment of neurodegenerative conditions. Certain embodiments encompass pharmaceutical compositions comprising live cells (UTCs alone or admixed with other cell types). Other embodiments encompass pharmaceutical compositions comprising UTC cellular components (e.g., cell lysates, soluble cell fractions, conditioned medium, ECM, or components of any of the foregoing) or products (e.g., trophic and other biological factors produced naturally by UTCs or through genetic modification, conditioned medium from UTC culture). In either case, the pharmaceutical composition may further comprise other active agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors or neuroregenerative or neuroprotective drugs as known in the art.

Examples of other components that may be added to UTC pharmaceutical compositions include, but are not limited to: (1) other neuroprotective or neurobeneficial drugs; (2) selected extracellular matrix components, such as one or more types of collagen known in the art, and/or growth factors, platelet-rich plasma, and drugs (alternatively, UTCs may be genetically engineered to express and produce growth factors); (3) anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocyte growth factor, caspase inhibitors); (4) anti-inflammatory compounds (e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE®, SIROLIMUS, and non-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPDXALIN, TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatory agents, such as calcineurin inhibitors, mTOR inhibitors, antiproliferatives, corticosteroids and various antibodies; (6) antioxidants such as probucol, vitamins C and E, conenzyme Q-10, glutathione, L-cysteine and N-acetylcysteine; (7) local anesthetics; and (8) neurotrophic factors such as GDF5, BMP-14, CDMP-1, MP52, BMP7, Sonic Hedgehog (SHH), Fibroblast Growth Factor 8 (FGF8), BDNF, NGF, to name a few.

Pharmaceutical compositions of the invention comprise UTCs, or components or products thereof, formulated with a pharmaceutically acceptable carrier or medium. Suitable pharmaceutically acceptable carriers include water, salt solution (such as Ringer's solution), alcohols, oils, gelatins, and carbohydrates, such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, and polyvinyl pyrrolidine. Such preparations can be sterilized, and if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring. Pharmaceutical carriers suitable for use in the present invention are known in the art and are described, for example, in Pharmaceutical Sciences (17^(th) Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309.

Typically, but not exclusively, pharmaceutical compositions comprising UTC components or products, but not live cells, are formulated as liquids (or as solid tablets, capsules and the like, when oral delivery is appropriate). These may be formulated for administration by any acceptable route known in the art to achieve delivery of drugs and biological molecules to the target neural tissue, including, but not limited to, oral, nasal, ophthalmic and parenteral, including intravenous. Particular routes of parenteral administration include, but are not limited to, intramuscular, subcutaneous, intraperitoneal, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal and/or peri-spinal routes of administration by delivery via intracranial or intravertebral needles and/or catheters with or without pump devices.

Pharmaceutical compositions comprising UTC live cells are typically formulated as liquids, semisolids (e.g., gels) or solids (e.g., matrices, scaffolds and the like, as appropriate for neural tissue engineering). Liquid compositions are formulated for administration by any acceptable route known in the art to achieve delivery of live cells to the target neural tissues. Typically, these include injection or infusion into the CNS or PNS, either in a diffuse fashion or targeted to the site of neurological disease or distress, by a route of administration including, but not limited to, intraocular, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal and/or peri-spinal routes of administration by delivery via intracranial or intravertebral needles and/or catheters with or without pump devices. When injected or infused, the calles may migrate to the site of the injury.

Pharmaceutical compositions comprising live cells in a semi-solid or solid carrier are typically formulated for surgical implantation at the site of neurological damage or distress. It will be appreciated that liquid compositions also may be administered by surgical procedures. In particular embodiments, semi-solid or solid pharmaceutical compositions may comprise semi-permeable gels, lattices, cellular scaffolds and the like, which may be non-biodegradable or biodegradable. For example, in certain embodiments, it may be desirable or appropriate to sequester the exogenous cells from their surroundings, yet enable the cells to secrete and deliver biological molecules (e.g. neurotrophic factors) to surrounding neural cells. In these embodiments, cells may be formulated as autonomous implants comprising living UTCs or cell population comprising UTCs surrounded by a non-degradable, selectively permeable barrier that physically separates the transplanted cells from host tissue. Such implants are sometimes referred to as “immunoprotective,” as they have the capacity to prevent immune cells and macromolecules from killing the transplanted cells in the absence of pharmacologically induced immunosuppression (for a review of such devices and methods, see, e.g., P. A. Tresco et al., 2000, Adv. Drug Delivery Rev. 42: 3-27).

In one embodiment of the invention, the pharmaceutical compositions comprising umbilical cord tissue-derived cells, the substantially homogenous populations comprising umbilical cord tissue-derived cells or the umbilical cord tissue-derived cells are administered by intravenous and/or intrathecal injection. In another embodiment, the pharmaceutical compositions comprising umbilical cord tissue-derived cells, the substantially homogenous populations comprising umbilical cord tissue-derived cells or the umbilical cord tissue-derived cells are repeatedly administered by intravenous and/or intrathecal injection.

In other embodiments, different varieties of degradable gels and networks are utilized for the pharmaceutical compositions of the invention. For example, degradable materials particularly suitable for sustained release formulations include biocompatible polymers, such as poly(lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including, A. Domb et al., 1992, Polymers for Advanced Technologies 3:279.

In other embodiments, e.g., for repair of large neural lesions, such as a damaged or severed spinal cord or a neural cord of a severed limb, it may be desirable or appropriate to deliver the cells on or in a biodegradable, preferably bioresorbable or bioabsorbable, scaffold or matrix. These typically three-dimensional biomaterials contain the living cells attached to the scaffold, dispersed within the scaffold, or incorporated in an extracellular matrix entrapped in the scaffold. Once implanted into the target region of the body, these implants become integrated with the host tissue, wherein the transplanted cells gradually become established (see, e.g., P. A. Tresco et al., 2000, supra; see also D. W. Hutmacher, 2001, J. Biomater. Sci. Polymer Edn. 12: 107-174).

The biocompatible matrix may be comprised of natural, modified natural or synthetic biodegradable polymers, including homopolymers, copolymers and block polymers, as well as combinations thereof. It is noted that a polymer is generally named based on the monomer from which it is synthesized.

Examples of suitable biodegradable polymers or polymer classes include fibrin, collagen, elastin, gelatin, vitronectin, fibronectin, laminin, reconstituted basement membrane matrices, starches, dextrans, alginates, hyaluron, chitin, chitosan, agarose, polysaccharides, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, their derivatives and mixtures thereof. For both glycolic acid and lactic acid, an intermediate cyclic dimer is typically prepared and purified prior to polymerization. These intermediate dimers are called glycolide and lactide, respectively. Other useful biodegradable polymers or polymer classes include, without limitation, polydioxanones, polycarbonates, polyoxalates, poly(alpha-esters), polyanhydrides, polyacetates, polycaprolactones, poly(orthoesters), polyamino acids, polyamides and mixtures and copolymers thereof. Additional useful biodegradable polymers include, without limitation stereopolymers of L- and D-lactic acid, copolymers of bis(para-carboxyphenoxy) propane acid and sebacic acid, sebacic acid copolymers, copolymers of caprolactone, poly(lactic acid)/poly(glycolic acid)/polyethyleneglycol copolymers, copolymers of polyurethane and poly(lactic acid), copolymers of polyurethane and poly(lactic acid), copolymers of alpha-amino acids, copolymers of alpha-amino acids and caproic acid, copolymers of alpha-benzyl glutamate and polyethylene glycol, copolymers of succinate and poly(glycols), polyphosphazene, polyhydroxy-alkanoates and mixtures thereof. Binary and ternary systems also are contemplated.

In general, a suitable biodegradable polymer for use as the matrix is desirably configured so that it has mechanical properties that are suitable for the intended application, remains sufficiently intact until tissue has in-grown and healed, does not invoke an inflammatory or toxic response, is metabolized in the body after fulfilling its purpose, is easily processed into the desired final product to be formed, demonstrates acceptable shelf-life, and is easily sterilized.

In one aspect of the invention, the biocompatible polymer used to form the matrix is in the form of a hydrogel. In general, hydrogels are cross-linked polymeric materials that can absorb more than 20% of their weight in water while maintaining a distinct three-dimensional structure. This definition includes dry cross-linked polymers that will swell in aqueous environments, as well as water-swollen materials. A host of hydrophilic polymers can be cross-linked to produce hydrogels, whether the polymer is of biological origin, semi-synthetic, or wholly synthetic. The hydrogel may be produced from a synthetic polymeric material. Such synthetic polymers can be tailored to a range of properties and predictable lot-to-lot uniformity, and represent a reliable source of material that generally is free from concerns of immunogenicity. The matrices may include hydrogels formed from self assembling peptides, as those discussed in U.S. Pat. Nos. 5,670,483 and 5,955,343, U.S. Patent Application No. 2002/0160471, PCT Application No. WO02/062969.

Properties that make hydrogels valuable in drug delivery applications include the equilibrium swelling degree, sorption kinetics, solute permeability, and their in vivo performance characteristics. Permeability to compounds depends in part upon the swelling degree or water content and the rate of biodegradation. Since the mechanical strength of a gel declines in direct proportion to the swelling degree, it is also well within the contemplation of the present invention that the hydrogel can be attached to a substrate so that the composite system enhances mechanical strength. In alternative embodiments, the hydrogel can be impregnated within a porous substrate, so as to gain the mechanical strength of the substrate, along with the useful delivery properties of the hydrogel.

Examples of scaffold or matrix (sometimes referred to collectively as “framework”) material that may be used in the present invention include nonwoven mats, porous foams, or self assembling peptides. Nonwoven mats may, for example, be formed using fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (PGA/PLA), sold under the trade name VICRYL® (Ethicon, Inc., Somerville, N.J.), Foams, composed of, for example, poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed by processes such as freeze-drying, or lyophilized, as discussed in U.S. Pat. No. 6,355,699, also may be utilized. Hydrogels such as self-assembling peptides (e.g., RAD16) may also be used. In situ-forming degradable networks are also suitable for use in the invention (see, e.g., Anseth, K. S. et al., 2002, J. Controlled Release 78: 199-209; Wang, D. et al., 2003, Biomaterials 24: 3969-3980; U.S. Patent Publication 2002/0022676 to He et al.). These materials are formulated as fluids suitable for injection, then may be induced by a variety of means (e.g., change in temperature, pH, exposure to light) to form degradable hydrogel networks in situ or in vivo.

In another embodiment, the framework is a felt, which can be composed of a multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA, PCL copolymers or blends, or hyaluronic acid. The yarn is made into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling. In another embodiment, cells are seeded onto foam scaffolds that may be composite structures.

In many of the abovementioned embodiments, the framework may be molded into a useful shape, such as that of the spinal cord with segregated columns for nerve tract repair, for example (Friedman J A et al., 2002, Neurosurgery 51: 742-51). Furthermore, it will be appreciated that UTCs may be cultured on pre-formed, non-degradable surgical or implantable devices, e.g., in a manner corresponding to that used for preparing fibroblast-containing GDC endovascular coils, for instance (Marx, W. F. et al., 2001, Am. J. Neuroradiol. 22: 323-333).

The matrix, scaffold or device may be treated prior to inoculation of cells in order to enhance cell attachment. For example, prior to inoculation, nylon matrices can be treated with 0.1 molar acetic acid and incubated in polylysine, PBS, and/or collagen to coat the nylon. Polystyrene can be similarly treated using sulfuric acid. The external surfaces of a framework may also be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma coating the framework or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellular matrix, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, among others.

UTC-containing frameworks are prepared according to methods known in the art. For example, cells can be grown freely in a culture vessel to sub-confluency or confluency, lifted from the culture and inoculated onto the framework. Growth factors may be added to the culture medium prior to, during, or subsequent to inoculation of the cells to trigger differentiation and tissue formation, if desired. Alternatively, the frameworks themselves may be modified so that the growth of cells thereon is enhanced, or so that the risk of rejection of the implant is reduced. Thus, one or more biologically active compounds, including, but not limited to, anti-inflammatories, immunosuppressants or growth factors, may be added to the framework for local release.

Methods of Using UTCs, UTC Components or Products

UTCs, or cell populations comprising UTCs, or components of or products produced by UTCs, may be used in a variety of ways to support and facilitate repair and regeneration of neural cells and tissues, and to improve neurological function and behavior. Such utilities encompass in vitro, ex vivo and in vivo methods. For example, UTCs, or cell populations comprising UTCs, or components of or products produced by UTCs, may be used to treat stroke.

In Vitro and Ex Vivo Methods:

In one embodiment, UTCs may be used in vitro to screen a wide variety of compounds for effectiveness and cytotoxicity of pharmaceutical agents, growth factors, regulatory factors, and the like. For example, such screening may be performed on substantially homogeneous populations of UTCs to assess the efficacy or toxicity of candidate compounds to be formulated with, or co-administered with, the UTCs, for treatment of a neurodegenerative condition. Alternatively, such screening may be performed on UTCs that have been stimulated to differentiate into a neural cell or neural progenitor cell, for the purpose of evaluating the efficacy of new pharmaceutical drug candidates. In this embodiment, the UTCs are maintained in vitro and exposed to the compound to be tested. The activity of a potentially cytotoxic compound can be measured by its ability to damage or kill cells in culture. This may readily be assessed by vital staining techniques. The effect of growth or regulatory factors may be assessed by analyzing the number or robustness of the cultured cells, as compared with cells not exposed to the factors. This may be accomplished using standard cytological and/or histological techniques, including the use of immunocytochemical techniques employing antibodies that define type-specific cellular antigens.

In a further embodiment, as discussed above, UTCs can be cultured in vitro to produce biological products that are either naturally produced by the cells, or produced by the cells when induced to differentiate into neural lineages, or produced by the cells via genetic modification. For instance, TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP 1beta, MCP1, RANTES, 1309, TARC, MDC, and IL-8 were found to be secreted from umbilicus-derived cells grown in Growth Medium. TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1alpha, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were found to be secreted from placenta-derived cells cultured in Growth Medium (see Examples). Some of these trophic factors, such as BDNF and IL-6, have important roles in neural regeneration. Other trophic factors, as yet undetected or unexamined, of use in neural repair and regeneration, are likely to be produced by UTCs and possibly secreted into the medium.

In this regard, another embodiment of the invention features use of UTCs for production of conditioned medium, either from undifferentiated UTCs or from UTCs incubated under conditions that stimulate differentiation into a neural lineage. Such conditioned media are contemplated for use in in vitro or ex vivo culture of neurogeneic precursor cells, or in vivo to support transplanted cells comprising homogeneous populations of UTCs or heterogeneous populations comprising UTCs and neural progenitors, for example.

Yet another embodiment comprises the use of UTC cell lysates, soluble cell fractions or components thereof, or ECM or components thereof, for a variety of purposes. As mentioned above, some of these components may be used in pharmaceutical compositions. In other embodiments, a cell lysate or ECM is used to coat or otherwise treat substances or devices to be used surgically, or for implantation, or for ex vivo purposes, to promote healing or survival of cells or tissues contacted in the course of such treatments.

As described in Examples 13 and 15, umbilicus and placenta derived-cells have demonstrated the ability to support survival, growth and differentiation of adult neural progenitor cells when grown in co-culture with those cells. Accordingly, in another embodiment, UTCs are used advantageously in co-cultures in vitro to provide trophic support to other cells, in particular neural cells and neural progenitors. For co-culture, it may be desirable for the UTCs and the desired other cells to be co-cultured under conditions in which the two cell types are in contact. This can be achieved, for example, by seeding the cells as a heterogeneous population of cells in culture medium or onto a suitable culture substrate. Alternatively, the UTCs can first be grown to confluence, and then will serve as a substrate for the second desired cell type in culture. In this latter embodiment, the cells may further be physically separated, e.g., by a membrane or similar device, such that the other cell type may be removed and used separately, following the co-culture period. Use of UTCs in co-culture to promote expansion and differentiation of neural cell types may find applicability in research and in clinical/therapeutic areas. For instance, UTC co-culture may be utilized to facilitate growth and differentiation of neural cells in culture, for basic research purposes or for use in drug screening assays, for example. UTC co-culture may also be utilized for ex vivo expansion of neural progenitors for later administration for therapeutic purposes. For example, neural progenitor cells may be harvested from an individual, expanded ex vivo in co-culture with UTCs, then returned to that individual (autologous transfer) or another individual (syngeneic or allogeneic transfer). In these embodiments, it will be appreciated that, following ex vivo expansion, the mixed population of cells comprising the UTCs and neural progenitors could be administered to a patient in need of treatment. Alternatively, in situations where autologous transfer is appropriate or desirable, the co-cultured cell populations may be physically separated in culture, enabling removal of the autologous neural progenitors for administration to the patient.

Alternatively, neuroprogenitor cells or astrocytes could be subjected to an ischemic insult to activate similar pathways to those occurring during stroke. Cells could also be pre-conditioned by incubation in ischemic conditions.

In Vivo Methods:

As set forth in Examples 16 to 19, 23 to 26, UTCs have been shown to be effectively transplanted into the body, and to supply lost neural function in an animal model accepted for its predictability of efficacy in humans. In particular, as shown in Examples 25 and 26, hUTC administered intranasally have been shown to be effectively transplanted into the body, and to supply lost neural function in an animal model of stroke accepted for its predictability of efficacy in humans. These results support preferred embodiments of the invention, wherein UTCs are used in cell therapy for treating strokes by repairing neural tissue in a stroke patient, wherein UTCs are used in cell therapy for treating a stroke by regenerating neural tissue in a stroke patient, and wherein UTCs are used in cell therapy for treating a stroke by improving neurological function or behavior in a stroke patient. In one embodiment, the UTCs are transplanted into a target neural location in the body, where the UTCs can differentiate into one or more neural phenotypes, or the UTCs may provide trophic support for neural progenitors and neural cells in situ, or the UTCs may exert a beneficial effect in both of those fashions, among others. In one preferred embodiment, the UTC are administered intranasally.

Specific embodiments of the invention are directed to support, regeneration or replacement of dopaminergic (DA) neurons and other conditions affecting DA-rich regions of the brain, e.g., strokes affecting the middle cerebral artery (MCA) and its branches. As described in detail in Example 18, UTCs were shown efficacious in the treatment of rats in which middle cerebral artery occlusion was induced temporally. In the cell efficacy study, statistically significant improvement was observed in behavioral tests; functional recovery was compared to control rats receiving either PBS or mesenchymal stem cells. In addition, transplanted UTCs were able to home to the site of ischemic injury from a systemic administration via tail vein injection. UTCs were observed to survive in the rat brain for the duration of the study (28 days). This was accompanied by indicators of neurotrophic benefits, including increases in neurogenesis, angiogenesis and synaptogenesis, as well as a lower incidence of cellular apoptosis and death, in the ischemic rat brain. Such benefits could be the result of UTCs stimulating host cell migration, proliferation, and neurogenesis. Remarkably, the transplantation of human UTCs into rats did not require usage of immunosuppressants.

UTCs may be administered alone (e.g., as substantially homogeneous populations) or as admixtures with other cells. As described above, UTCs may be administered as formulated in a pharmaceutical preparation with a matrix or scaffold, or with conventional pharmaceutically acceptable carriers. Where UTCs are administered with other cells, they may be administered simultaneously or sequentially with the other cells (either before or after the other cells). Cells that may be administered in conjunction with UTCs include, but are not limited to, neurons, astrocytes, oligodendrocytes, neural progenitor cells, neural stem cells and/or other multipotent or pluripotent stem cells. The cells of different types may be admixed with the UTCs immediately or shortly prior to administration, or they may be co-cultured together for a period of time prior to administration. UTCs may be subjected to short periods of hypoxia prior to administration, to increase their chances of survival and/or production of beneficial factors. The potassium channels of UTCs can be modulated via one to multiple short bursts of exposure to modulating drugs.

The UTCs may be administered with other neuro-beneficial drugs or biological molecules, or other active agents, such as anti-inflammatory agents, anti-apoptotic agents, antioxidants, growth factors, neurotrophic factors or neuroregenerative or neuroprotective drugs as known in the art. When UTCs are administered with other agents, they may be administered together in a single pharmaceutical composition, or in separate pharmaceutical compositions, simultaneously or sequentially with the other agents (either before or after administration of the other agents). The other agents may be a part of a treatment regimen that begins either before transplantation and continuing throughout the course of recovery, or may be initiated at the time of transplantation, or even after transplantation, as a physician of skill in the art deems appropriate.

Examples of other components that may be administered with UTCs include, but are not limited to: (1) other neuroprotective or neurobeneficial drugs; (2) selected extracellular matrix components, such as one or more types of collagen known in the art, and/or growth factors, platelet-rich plasma, and drugs (alternatively, UTCs may be genetically engineered to express and produce growth factors); (3) anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocyte growth factor, caspase inhibitors); (4) anti-inflammatory compounds (e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE®, SIROLIMUS, and non-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPDXALIN, TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatory agents, such as calcineurin inhibitors, mTOR inhibitors, antiproliferatives, corticosteroids and various antibodies; (6) antioxidants such as probucol, vitamins C and E, conenzyme Q-10, glutathione, L-cysteine and N-acetylcysteine; and (7) local anesthetics, to name a few.

For in vivo use, the hUTC may also be pre-treated with suitable agent such as hyaluronidase. In one embodiment, the hUTC are co-administered with an immunosuppressive agent such as e.g. cyclosporine.

One preferred embodiment of the invention is the treatment of stroke with hUTC that are administered intranasally. The hUTC may be administered as a component of a pharmaceutical composition specifically formulated for intranasal administration. In one embodiment, 1×10⁶ hUTC cells are administered intranasally for the treatment of stroke. In certain embodiments, the hUTC are co-administered with an enzyme inhibitor or an absorption enhancer. In other embodiments, the pharmaceutical compositions formulated for intranasal administration comprise enzyme inhibitor and/or absorption enhancers. In yet other embodiments, the pharmaceutical compositions comprise synthetic surfactants, bile salts, phospholipids, and cylodextrins. The hUTC may also be intranasally administered via an emulsion or a liposome. In certain embodiments, intranasal administration is achieved by use of polymeric microspheres. The hUTC may be administered in the presence of sodium glycohcholate (NaGC) and linoleic acid.

The pharmaceutical composition for intranasal administration may be administered as a spray, aerosol, gel, solution, emulsion, or suspension. Alternatively, the pharmaceutical composition is administered directly to the upper airways such as e.g. the paranasal sinuses. In one embodiment, the hUTC or the pharmaceutical composition are administered via a microcatheter.

In one embodiment, UTCs are administered as undifferentiated cells, i.e., as cultured in Growth Medium. Alternatively, UTCs may be administered following exposure in culture to conditions that stimulate differentiation toward a desired neural phenotype, e.g., astrocyte, oligodendrocyte or neuron, and more specifically, serotoninergic, dopaminergic, cholinergic, GABA-ergic or glutamatergic neurons (see, e.g., Isacson, O., 2003. The Lancet (Neurology) 2: 417-424).

The cells of the invention may be surgically implanted, injected, delivered (e.g., by way of a catheter or syringe), or otherwise administered directly or indirectly to the site of neurological damage or distress. Routes of administration of the cells of the invention or compositions thereof include, but are not limited to, intravenous, intramuscular, subcutaneous, intranasal, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal and/or peri-spinal routes of administration by delivery via intracranial or intravertebral needles and/or catheters with or without pump devices.

When cells are administered in semi-solid or solid devices, surgical implantation into a precise location in the body is typically a suitable means of administration. Liquid or fluid pharmaceutical compositions, however, may be administered to a more general location in the CNS or PNS (e.g., throughout a diffusely affected area, such as would be expected for a diffuse ischemic injury), inasmuch as neural progenitor cells have been shown to be capable of extensive migration from a point of entry to the nervous system to a particular location, e.g., by following radial glia or by responding to chemical signals.

The umbilical-derived cells or compositions and/or matrices comprising the umbilical-derived cells may be delivered to the site via a micro catheter, intracatheterization, shunt, cannula, or via a mini-pump. The compositions and/or matrices could also be indirectly delivered to the substantia nigra or striatum via intrathecal delivery, or intracerebroventricularly, or by intranasal administration. The vehicle excipient or carrier can be any of those known to be pharmaceutically acceptable for administration to a patient, particularly locally at the site at which cellular differentiation is to be induced. Examples include liquid media, for example, Dulbeccos Modified Eagle's Medium (DMEM), sterile saline, sterile phosphate buffered saline, Leibovitz's medium (L15, Invitrogen, Carlsbad, Calif.), dextrose in sterile water, and any other physiologically acceptable liquid.

A method of delivery into the damaged region is intrathecally or intracerebroventricularly with, for example, an Ommaya reservoir in accordance with known techniques such as those taught in F. Balis and D. Poplack (1989) Am. J. Pediatric. Hematol. Oncol. 11(1):74-86. An even more preferred method of delivery into the damaged region is by direct intraparenchymal injection via a microcatheter.

Indeed, this migratory ability of neural stem cells has opened a new avenue for treatment of malignant brain tumors, i.e., use of progenitor cells for delivery of therapeutic genes/gene products for the treatment of these migratory tumors. For example, it has been reported that neural stem cells, when implanted into intracranial gliomas in vivo in adult rodents, distribute themselves quickly and extensively through the tumor bed and migrate in juxtaposition to expanding and advancing tumor cells, while continuing to stably express a foreign gene (Aboody, K. et al., 2000, Proc. Natl. Acad. Sci. USA 97: 12846-12851). UTCs are also expected to be suitable for this type of use, i.e., UTCs genetically modified to produce an apoptotic or other antineoplastic agent, e.g., IL-12 (Ehtesham, M. et al., 2002, Cancer Research 62: 5657-5663) or tumor necrosis factor-related apoptosis-inducing ligand (Ehtesham, M. et al., 2002, Cancer Research 62: 7170-7174) may be injected or otherwise administered to a general site of a malignant tumor (e.g., glioblastoma), whereafter the UTCs can migrate to the tumor cells for local delivery of the therapeutic agent.

Other embodiments encompass methods of treating neurodegenerative conditions by administering pharmaceutical compositions comprising UTC cellular components (e.g., cell lysates or components thereof) or products (e.g., trophic and other biological factors produced naturally by UTCs or through genetic modification, conditioned medium from UTC culture). Again, these methods may further comprise administering other active agents, such as growth factors, neurotrophic factors or neuroregenerative or neuroprotective drugs as known in the art.

Dosage forms and regimes for administering UTCs or any of the other pharmaceutical compositions described herein are developed in accordance with good medical practice, taking into account the condition of the individual patient, e.g., nature and extent of the neurodegenerative condition, age, sex, body weight and general medical condition, and other factors known to medical practitioners. Thus, the effective amount of a pharmaceutical composition to be administered to a patient is determined by these considerations as known in the art.

Because the CNS is a somewhat immunoprivileged tissue, it may not be necessary or desirable to immunosuppress a patient prior to initiation of cell therapy with UTCs. In addition, as set forth in Example 11, UTCs have been shown not to stimulate allogeneic PBMCs in a mixed lymphocyte reaction. Accordingly, transplantation with allogeneic, or even xenogeneic, UTCs may be tolerated in some instances.

However, in other instances it may be desirable or appropriate to pharmacologically immunosuppress a patient prior to initiating cell therapy. This may be accomplished through the use of systemic or local immunosuppressive agents, or it may be accomplished by delivering the cells in an encapsulated device, as described above. These and other means for reducing or eliminating an immune response to the transplanted cells are known in the art. As an alternative, UTCs may be genetically modified to reduce their immunogenicity, as mentioned above.

Survival of transplanted UTCs in a living patient can be determined through the use of a variety of scanning techniques, e.g., computerized axial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) or positron emission tomography (PET) scans. Determination of transplant survival can also be done post mortem by removing the neural tissue, and examining it visually or through a microscope. Alternatively, cells can be treated with stains that are specific for neural cells or products thereof, e.g., neurotransmitters. Transplanted cells can also be identified by prior incorporation of tracer dyes such as rhodamine- or fluorescein-labeled microspheres, fast blue, ferric microparticles, bisbenzamide or genetically introduced reporter gene products, such as beta-galactosidase or beta-glucuronidase.

Functional integration of transplanted UTCs into neural tissue of a subject can be assessed by examining restoration of the neural function that was damaged or diseased. Such functions include, but are not limited to motor, cognitive, sensory and endocrine functions, in accordance with procedures well known to neurobiologists and physicians.

Kits and Banks Comprising UTCs, UTC Components or Products

In another aspect, the invention provides kits that utilize the UTCs, UTC populations, components and products of UTCs in various methods for neural regeneration and repair as described above. Where used for treatment of neurodegenerative conditions, or other scheduled treatment, the kits may include one or more cell populations, including at least UTCs and a pharmaceutically acceptable carrier (liquid, semi-solid or solid). The kits also optionally may include a means of administering the cells, for example by injection. The kits further may include instructions for use of the cells. Kits prepared for field hospital use, such as for military use may include full-procedure supplies including tissue scaffolds, surgical sutures, and the like, where the cells are to be used in conjunction with repair of acute injuries. Kits for assays and in vitro methods as described herein may contain one or more of (1) UTCs or components or products of UTCs, (2) reagents for practicing the in vitro method, (3) other cells or cell populations, as appropriate, and (4) instructions for conducting the in vitro method.

In yet another aspect, the invention also provides for banking of tissues, cells, cellular components and cell populations of the invention. As discussed above, the cells are readily cryopreserved. The invention therefore provides methods of cryopreserving the cells in a bank, wherein the cells are stored frozen and associated with a complete characterization of the cells based on immunological, biochemical and genetic properties of the cells. The frozen cells can be thawed and expanded or used directly for autologous, syngeneic, or allogeneic therapy, depending on the requirements of the procedure and the needs of the patient. Preferably, the information on each cryopreserved sample is stored in a computer, which is searchable based on the requirements of the surgeon, procedure and patient with suitable matches being made based on the characterization of the cells or populations. Preferably, the cells of the invention are grown and expanded to the desired quantity of cells and therapeutic cell compositions are prepared either separately or as co-cultures, in the presence or absence of a matrix or support. While for some applications it may be preferable to use cells freshly prepared, the remainder can be cryopreserved and banked by freezing the cells and entering the information in the computer to associate the computer entry with the samples. Even where it is not necessary to match a source or donor with a recipient of such cells, for immunological purposes, the bank system makes it easy to match, for example, desirable biochemical or genetic properties of the banked cells to the therapeutic needs. Upon matching of the desired properties with a banked sample, the sample is retrieved and prepared for therapeutic use. Cell lysates, ECM or cellular components prepared as described herein may also be cryopreserved or otherwise preserved (e.g., by lyophilization) and banked in accordance with the present invention.

The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.

As used in the following examples and elsewhere in the specification, the term Growth Medium generally refers to a medium sufficient for the culturing of UTCs. In particular, one presently preferred medium for the culturing of the cells of the invention in comprises Dulbecco's Modified Essential Media (also abbreviated DMEM herein). Particularly preferred is DMEM-low glucose (also DMEM-LG herein) (Invitrogen, Carlsbad, Calif.). The DMEM-low glucose is preferably supplemented with 15% (v/v) fetal bovine serum (e.g. defined fetal bovine serum, Hyclone, Logan Utah), antibiotics/antimycotics ((preferably 50-100 Units/milliliter penicillin, 50-100 microgram/milliliter streptomycin, and 0-0.25 microgram/milliliter amphotericin B; Invitrogen, Carlsbad, Calif.)), and 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.). As used in the Examples below, Growth Medium refers to DMEM-low glucose with 15% fetal bovine serum and antibiotics/antimycotics (when penicillin/streptomycin are included, it is preferably at 50 U/milliliter and 50 microgram/milliliter respectively; when penicillin/streptomycin/amphotericin B are use, it is preferably at 100 U/milliliter, 100 microgram/milliliter and 0.25 microgram/milliliter, respectively). In some cases different growth media are used, or different supplementations are provided, and these are normally indicated in the text as supplementations to Growth Medium.

Also relating to the following examples and used elsewhere in the specification, the term standard growth conditions refers to culturing of cells at 37° C., in a standard atmosphere comprising 5% CO₂. While foregoing the conditions are useful for culturing, it is to be understood that such conditions are capable of being varied by the skilled artisan who will appreciate the options available in the art for culturing cells.

The following abbreviations may appear in the examples and elsewhere in the specification and claims: ANG2 (or Ang2) for angiopoietin 2; APC for antigen-presenting cells; BDNF for brain-derived neurotrophic factor; bFGF for basic fibroblast growth factor; bid (BID) for “bis in die” (twice per day); CK18 for cytokeratin 18; CNS for central nervous system; CXC ligand 3 for chemokine receptor ligand 3; DMEM for Dulbecco's Minimal Essential Medium; DMEM:lg (or DMEM:Lg, DMEM:LG) for DMEM with low glucose; EDTA for ethylene diamine tetraacetic acid; EGF (or E) for epidermal growth factor; FACS for fluorescent activated cell sorting; FBS for fetal bovine serum; FGF (or F) for fibroblast growth factor; GCP-2 for granulocyte chemotactic protein-2; GFAP for glial fibrillary acidic protein; HB-EGF for heparin-binding epidermal growth factor; HCAEC for Human coronary artery endothelial cells; HGF for hepatocyte growth factor; hMSC for Human mesenchymal stem cells; HNF-1alpha for hepatocyte-specific transcription factor 1 alpha; HUVEC for Human umbilical vein endothelial cells; 1309 for a chemokine and the ligand for the CCR8 receptor; IGF-1 for insulin-like growth factor 1; IL-6 for interleukin-6; IL-8 for interleukin 8; K19 for keratin 19; K8 for keratin 8; KGF for keratinocyte growth factor; LIF for leukemia inhibitory factor; MBP for myelin basic protein; MCP-1 for monocyte chemotactic protein 1; MDC for macrophage-derived chemokine; MIP 1 alpha for macrophage inflammatory protein 1 alpha; MIP1beta for macrophage inflammatory protein 1 beta; MMP for matrix metalloprotease (MMP); MSC for mesenchymal stem cells; NHDF for Normal Human Dermal Fibroblasts; NPE for Neural Progenitor Expansion media; O4 for oligodendrocyte or glial differentiation marker O4; PBMC for Peripheral blood mononuclear cell; PBS for phosphate buffered saline; PDGFbb for platelet derived growth factor; PO for “per os” (by mouth); PNS for peripheral nervous system; Rantes (or RANTES) for regulated on activation, normal T cell expressed and secreted; rhGDF-5 for recombinant human growth and differentiation factor 5; SC for subcutaneously; SDF-1 alpha for stromal-derived factor 1 alpha; SHH for sonic hedgehog; SOP for standard operating procedure; TARC for thymus and activation-regulated chemokine; TCP for Tissue culture plastic; TCPS for tissue culture polystyrene; TGFbeta2 for transforming growth factor beta2; TGF beta-3 for transforming growth factor beta-3; TIMP1 for tissue inhibitor of matrix metalloproteinase 1; TPO for thrombopoietin; TuJ1 for BIII Tubulin; VEGF for vascular endothelial growth factor; νWF for von Willebrand factor; and alphaFP for alpha-fetoprotein.

Example 1 Derivation of Cells from Umbilicus and Placenta Tissue

This example describes the preparation cells from placental and umbilical cord tissues. Umbilical cords and placentae were obtained upon birth of either a full term or pre-term pregnancy. Cells were harvested from 5 separate donors of umbilicus and placental tissue. Different methods of cell isolation were tested for their ability to yield cells with: 1) the potential to differentiate into cells with different phenotypes, a characteristic common to stem cells, or 2) the potential to provide trophic factors useful for other cells and tissues.

Methods & Materials

Umbilical cell isolation. Umbilical cords were obtained from National Disease Research Interchange (NDRI, Philadelphia, Pa.). The tissues were obtained following normal deliveries. The cell isolation protocol was performed aseptically in a laminar flow hood. To remove blood and debris, the cord was washed in phosphate buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in the presence of antimycotic and antibiotic (100 units/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B). The tissues were then mechanically dissociated in 150 cm² tissue culture plates in the presence of 50 milliliters of medium (DMEM-Low glucose or DMEM-High glucose; Invitrogen), until the tissue was minced into a fine pulp. The chopped tissues were transferred to 50-milliliter conical tubes (approximately 5 grams of tissue per tube). The tissue was then digested in either DMEM-Low glucose medium or DMEM-High glucose medium, each containing antimycotic and antibiotic as described above. In some experiments, an enzyme mixture of collagenase and dispase was used (“C:D;” collagenase (Sigma, St Louis, Mo.), 500 Units/milliliter; and dispase (Invitrogen), 50 Units/milliliter in DMEM:-Low glucose medium). In other experiments, a mixture of collagenase, dispase and hyaluronidase (“C:D:H”) was used (collagenase, 500 Units/milliliter; dispase, 50 Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter, in DMEM:-Low glucose). The conical tubes containing the tissue, medium and digestion enzymes were incubated at 37° C. in an orbital shaker (Environ, Brooklyn, N.Y.) at 225 rpm for 2 hrs.

After digestion, the tissues were centrifuged at 150×g for 5 minutes, the supernatant was aspirated. The pellet was resuspended in 20 milliliters of Growth Medium (DMEM:Low glucose (Invitrogen), 15 percent (v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475; Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma), 1 milliliter per 100 milliliters of antibiotic/antimycotic as described above. The cell suspension was filtered through a 70-micrometer nylon cell strainer (BD Biosciences). An additional 5 milliliters rinse comprising Growth Medium was passed through the strainer. The cell suspension was then passed through a 40-micrometer nylon cell strainer (BD Biosciences) and chased with a rinse of an additional 5 milliliters of Growth Medium.

The filtrate was resuspended in Growth Medium (total volume 50 milliliters) and centrifuged at 150×g for 5 minutes. The supernatant was aspirated and the cells were resuspended in 50 milliliters of fresh Growth Medium. This process was repeated twice more.

Upon the final centrifugation supernatant was aspirated and the cell pellet was resuspended in 5 milliliters of fresh Growth Medium. The number of viable cells was determined using Trypan Blue staining Cells were then cultured under standard conditions.

The cells isolated from umbilical cords were seeded at 5,000 cells/cm² onto gelatin-coated T-75 cm² flasks (Corning Inc., Corning, N.Y.) in Growth Medium with antibiotics/antimycotics as described above. After 2 days (in various experiments, cells were incubated from 2-4 days), spent medium was aspirated from the flasks. Cells were washed with PBS three times to remove debris and blood-derived cells. Cells were then replenished with Growth Medium and allowed to grow to confluence (about 10 days from passage 0) to passage 1. On subsequent passages (from passage 1 to 2 and so on), cells reached sub-confluence (75-85 percent confluence) in 4-5 days. For these subsequent passages, cells were seeded at 5000 cells/cm². Cells were grown in a humidified incubator with 5 percent carbon dioxide and atmospheric oxygen, at 37° C.

Placental Cell Isolation. Placental tissue was obtained from NDRI (Philadelphia, Pa.). The tissues were from a pregnancy and were obtained at the time of a normal surgical delivery. Placental cells were isolated as described for umbilical cell isolation.

The following example applies to the isolation of separate populations of maternal-derived and neonatal-derived cells from placental tissue.

The cell isolation protocol was performed aseptically in a laminar flow hood. The placental tissue was washed in phosphate buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in the presence of antimycotic and antibiotic (as described above) to remove blood and debris. The placental tissue was then dissected into three sections: top-line (neonatal side or aspect), mid-line (mixed cell isolation neonatal and maternal) and bottom line (maternal side or aspect).

The separated sections were individually washed several times in PBS with antibiotic/antimycotic to further remove blood and debris. Each section was then mechanically dissociated in 150 cm² tissue culture plates in the presence of 50 milliliters of DMEM/Low glucose, to a fine pulp. The pulp was transferred to 50 milliliter conical tubes. Each tube contained approximately 5 grams of tissue. The tissue was digested in either DMEM-Low glucose or DMEM-High glucose medium containing antimycotic and antibiotic (100 U/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B) and digestion enzymes. In some experiments an enzyme mixture of collagenase and dispase (“C:D”) was used containing collagenase (Sigma, St Louis, Mo.) at 500 Units/milliliter and dispase (Invitrogen) at 50 Units/milliliter in DMEM-Low glucose medium. In other experiments a mixture of collagenase, dispase and hyaluronidase (C:D:H) was used (collagenase, 500 Units/milliliter; dispase, 50 Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter in DMEM-Low glucose). The conical tubes containing the tissue, medium, and digestion enzymes were incubated for 2 h at 37° C. in an orbital shaker (Environ, Brooklyn, N.Y.) at 225 rpm.

After digestion, the tissues were centrifuged at 150×g for 5 minutes, the resultant supernatant was aspirated off. The pellet was resuspended in 20 milliliters of Growth Medium with penicillin/streptomycin/amphotericin B. The cell suspension was filtered through a 70 micrometer nylon cell strainer (BD Biosciences), chased by a rinse with an additional 5 milliliters of Growth Medium. The total cell suspension was passed through a 40 micrometer nylon cell strainer (BD Biosciences) followed with an additional 5 milliliters of Growth Medium as a rinse.

The filtrate was resuspended in Growth Medium (total volume 50 milliliters) and centrifuged at 150×g for 5 minutes. The supernatant was aspirated and the cell pellet was resuspended in 50 milliliters of fresh Growth Medium. This process was repeated twice more. After the final centrifugation, supernatant was aspirated and the cell pellet was resuspended in 5 milliliters of fresh Growth Medium. A cell count was determined using the Trypan Blue Exclusion test. Cells were then cultured at standard conditions.

LIBERASE™ Cell Isolation. Cells were isolated from umbilicus tissues in DMEM-Low glucose medium with LIBERASE™ (Boehringer Mannheim Corp., Indianapolis, Ind.) (2.5 milligrams per milliliter, Blendzyme 3; Roche Applied Sciences, Indianapolis, Ind.) and hyaluronidase (5 Units/milliliter, Sigma). Digestion of the tissue and isolation of the cells was as described for other protease digestions above, using the LIBERASE™/hyaluronidase mixture in place of the C:D or C:D:H enzyme mixture. Tissue digestion with LIBERASE™ resulted in the isolation of cell populations from umbilicus and placental tissues that expanded readily.

Cell isolation using other enzyme combinations. Procedures were compared for isolating cells from the umbilical cord using differing enzyme combinations. Enzymes compared for digestion included: i) collagenase; ii) dispase; iii) hyaluronidase; iv) collagenase:dispase mixture (C;D); v) collagenase:hyaluronidase mixture (C:H); vi) dispase:hyaluronidase mixture (D:H); and vii) collagenase:dispase:hyaluronidase mixture (C:D:H). Differences in cell isolation utilizing these different enzyme digestion conditions were observed (Table 1-1).

TABLE 1-1 Isolation of cells from umbilical cord tissue using varying enzyme combinations Cells Cell Enzyme Digest Isolated Expansion Collagenase X X Dispase + (>10 h) + Hyaluronidase X X Collagenase: Dispase ++ (<3 h) ++ Collagenase: Hyaluronidase ++ (<3 h) + Dispase: Hyaluronidase + (>10 h) + Collagenase: Dispase: Hyaluronidase +++ (<3 h) +++ Key: + = good, ++ = very good, +++ = excellent, X = no success under conditions tested

Isolation of cells from residual blood in the cords. Other attempts were made to isolate pools of cells from umbilical cord by different approaches. In one instance umbilical cord was sliced and washed with Growth Medium to dislodge the blood clots and gelatinous material. The mixture of blood, gelatinous material and Growth Medium was collected and centrifuged at 150×g. The pellet was resuspended and seeded onto gelatin-coated flasks in Growth Medium. From these experiments a cell population was isolated that readily expanded.

Isolation of cells from cord blood. Cells have also been isolated from cord blood samples attained from NDRI. The isolation protocol used here was that of International Patent Application PCT/US2002/029971 by Ho et al. (Ho, T. W. et al., WO2003025149 A2). Samples (50 milliliter and 10.5 milliliters, respectively) of umbilical cord blood (NDRI, Philadelphia Pa.) were mixed with lysis buffer (filter-sterilized 155 mM ammonium chloride, 10 millimolar potassium bicarbonate, 0.1 millimolar EDTA buffered to pH 7.2 (all components from Sigma, St. Louis, Mo.)). Cells were lysed at a ratio of 1:20 cord blood to lysis buffer. The resulting cell suspension was vortexed for 5 seconds, and incubated for 2 minutes at ambient temperature. The lysate was centrifuged (10 minutes at 200×g). The cell pellet was resuspended in complete minimal essential medium (Gibco, Carlsbad Calif.) containing 10 percent fetal bovine serum (Hyclone, Logan Utah), 4 millimolar glutamine (Mediatech Herndon, Va.), 100 Units penicillin per 100 milliliters and 100 micrograms streptomycin per 100 milliliters (Gibco, Carlsbad, Calif.). The resuspended cells were centrifuged (10 minutes at 200×g), the supernatant was aspirated, and the cell pellet was washed in complete medium. Cells were seeded directly into either T75 flasks (Corning, N.Y.), T75 laminin-coated flasks, or T175 fibronectin-coated flasks (both Becton Dickinson, Bedford, Mass.).

Isolation of cells using different enzyme combinations and growth conditions. To determine whether cell populations could be isolated under different conditions and expanded under a variety of conditions immediately after isolation, cells were digested in Growth Medium with or without 0.001 percent (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.), using the enzyme combination of C:D:H, according to the procedures provided above. Placental-derived cells so isolated were seeded under a variety of conditions. All cells were grown in the presence of penicillin/streptomycin. (Table 1-2).

TABLE 1-2 Isolation and culture expansion of cells under varying conditions: Condition Medium 15% FBS BME Gelatin 20% O₂ Growth Factors 1 DMEM-Lg Y Y Y Y N 2 DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg Y Y N Y N 4 DMEM-Lg Y Y N N (5%) N 5 DMEM-Lg N (2%) Y N (Laminin) Y EGF/FGF (20 ng/mL) 6 DMEM-Lg N (2%) Y N (Laminin) N (5%) EGF/FGF (20 ng/mL) 7 DMEM-Lg N (2%) Y N (Fibrone) Y PDGF/VEGF 8 DMEM-Lg N (2%) Y N (Fibrone) N (5%) PDGF/VEGF 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N (5%) N 11 DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%) N N (Laminin) Y EGF/FGF (20 ng/mL) 14 DMEM-Lg N (2%) N N (Laminin) N (5%) EGF/FGF (20 ng/mL) 15 DMEM-Lg N (2%) N N (Fibrone) Y PDGF/VEGF 16 DMEM-Lg N (2%) N N (Fibrone) N (5%) PDGF/VEGF

Isolation of cells using different enzyme combinations and growth conditions. In all conditions cells attached and expanded well between passage 0 and 1 (Table 1-2). Cells in conditions 5-8 and 13-16 were demonstrated to proliferate well up to 4 passages after seeding at which point they were cryopreserved and banked.

Results

Cell isolation using different enzyme combinations. The combination of C:D:H, provided the best cell yield following isolation, and generated cells which expanded for many more generations in culture than the other conditions (Table 1). An expandable cell population was not attained using collagenase or hyaluronidase alone. No attempt was made to determine if this result is specific to the collagen that was tested.

Isolation of cells using different enzyme combinations and growth conditions. Cells attached and expanded well between passage 0 and 1 under all conditions tested for enzyme digestion and growth (Table 2). Cells in experimental conditions 5-8 and 13-16 proliferated well up to 4 passages after seeding, at which point they were cryopreserved. All cells were banked for further investigation.

Isolation of cells from residual blood in the cords. Nucleated cells attached and grew rapidly. These cells were analyzed by flow cytometry and were similar to cells obtained by enzyme digestion.

Isolation of cells from cord blood. The preparations contained red blood cells and platelets. No nucleated cells attached and divided during the first 3 weeks. The medium was changed 3 weeks after seeding and no cells were observed to attach and grow.

Summary. Populations of cells can be derived from umbilical cord and placental tissue efficiently using the enzyme combination collagenase (a matrix metalloprotease), dispase (a neutral protease) and hyaluronidase (a mucolytic enzyme that breaks down hyaluronic acid). LIBERASE™, which is a Blendzyme, may also be used. Specifically, Blendzyme 3, which is collagenase (4 Wunsch units/g) and thermolysin (1714 casein Units/g) was also used together with hyaluronidase to isolate cells. These cells expanded readily over many passages when cultured in Growth Medium on gelatin coated plastic.

Cells were also isolated from residual blood in the cords, but not cord blood. The presence of cells in blood clots washed from the tissue, which adhere and grow under the conditions used, may be due to cells being released during the dissection process.

Example 2 Growth Characteristics of Umbilicus and Placenta-Derived Cells

The cell expansion potential of umbilical cord tissue and placental-derived cells was compared to other populations of isolated stem cells. The process of cell expansion to senescence is referred to as Hayflick's limit (Hayflick L. (1974) J Am Geriatr Soc. 22:1-12; Hayflick L. (1974) Gerontologist. 14:37-45). Umbilical cord tissue-derived cells are highly suited for therapeutic use because they can be readily expanded to sufficient cell numbers.

Materials and Methods

Gelatin-coating flasks. Tissue culture plastic flasks were coated by adding 20 milliliters 2% (w/v) porcine gelatin (Type B: 225 Bloom; Sigma, St Louis, Mo.) to a T75 flask (Corning, Corning, N.Y.) for 20 minutes at room temperature. After removing the gelatin solution, 10 milliliters phosphate-buffered saline (PBS) (Invitrogen, Carlsbad, Calif.) was added and then aspirated.

Comparison of expansion potential of UTCs with other cell populations. For comparison of growth expansion potential the following cell populations were utilized; i) Mesenchymal stem cells (MSC; Cambrex, Walkersville, Md.); ii) Adipose-derived cells (U.S. Pat. No. 6,555,374 B1; U.S. Pub. App. 2004/0058412); iii) Normal dermal skin fibroblasts (cc-2509 lot # 9F0844; Cambrex, Walkersville, Md.); iv) Umbilicus-derived cells; and v) Placenta-derived cells (U.S. Pub App. 2004/0048372). Cells were initially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks in Growth Medium with penicillin/streptomycin/amphotericin B. For subsequent passages, cell cultures were treated as follows. After trypsinization, viable cells were counted after Trypan Blue staining Cell suspension (50 microliters) was combined with Trypan Blue (50 milliliters, Sigma, St. Louis Mo.). Viable cell numbers were estimated using a hemocytometer.

Following counting, cells were seeded at 5,000 cells/cm² onto gelatin-coated T 75 flasks in 25 milliliters of fresh Growth Medium. Cells were grown under standard conditions at 37° C. The Growth Medium was changed twice per week. When cells reached about 85 percent confluence they were passaged; this process was repeated until the cells reached senescence.

At each passage, cells were trypsinized and counted. The viable cell yield, population doubling (ln(cell final/cell initial)/ln 2) and doubling time (time in culture (h)/population doubling) were calculated. For the purposes of determining optimal cell expansion, the total cell yield per passage was determined by multiplying the total yield for the previous passage by the expansion factor for each passage (i.e., expansion factor=cell final/cell initial).

Expansion potential of cell banks at low density. The expansion potential of cells banked at passage 10 was also tested, using a different set of conditions. Normal dermal skin fibroblasts (cc-2509 lot # 9F0844; Cambrex, Walkersville, Md.), umbilicus-derived cells, and placenta-derived cells were tested. These cell populations had been banked at passage 10 previously, having been cultured at 5,000 cells/cm² and grown to confluence at each passage to that point. The effect of cell density on the cell populations following cell thaw at passage 10 was determined. Cells were thawed under standard conditions and counted using Trypan Blue staining Thawed cells were then seeded at 1000 cells/cm² in DMEM:Low glucose Growth Medium with antibiotic/antimycotic as described above. Cells were grown under standard atmospheric conditions at 37° C. Growth Medium was changed twice a week and cells were passaged as they reached about 85% confluence. Cells were subsequently passaged until senescence, i.e., until they could not be expanded any further. Cells were trypsinized and counted at each passage. The cell yield, population doubling (ln(cell final/cell initial)/ln 2) and doubling time (time in culture (h)/population doubling). The total cell yield per passage was determined by multiplying total yield for the previous passage by the expansion factor for each passage (i.e., expansion factor=cell final/cell initial).

Expansion of UTCs at low density from initial cell seeding. The expansion potential of freshly isolated UTCs under low cell seeding conditions was tested. UTCs were prepared as described herein. Cells were seeded at 1000 cells/cm² and passaged as described above until senescence. Cells were grown under standard atmospheric conditions at 37° C. Growth Medium was changed twice per week. Cells were passaged as they reached about 85% confluence. At each passage, cells were trypsinized and counted by Trypan Blue staining. The cell yield, population doubling (ln(cell final/cell initial)/ln 2) and doubling time (time in culture (h)/population doubling) were calculated for each passage. The total cell yield per passage was determined by multiplying the total yield for the previous passage by the expansion factor for each passage (i.e. expansion factor=cell final/cell initial). Cells were grown on gelatin and non-gelatin coated flasks.

Expansion of clonal neonatal placenta-derived cells. Cloning was used in order to expand a population of neonatal cells from placental tissue. Following isolation of three differential cell populations from the placenta (as described herein), these cell populations were expanded under standard growth conditions and then karyotyped to reveal the identity of the isolated cell populations. Because the cells were isolated from a mother who delivered a boy, it was straightforward to distinguish between the male and female chromosomes by performing metaphase spreads. These experiments demonstrated that fetal-aspect cells were karyotype positive for neonatal phenotype, mid-layer cells were karyotype positive for both neonatal and maternal phenotypes and maternal-aspect cells were karyotype positive for maternal cells.

Expansion of cells in low oxygen culture conditions. It has been demonstrated that low oxygen cell culture conditions can improve cell expansion in certain circumstances (U.S. Pub. App. 2004/0005704). To determine if cell expansion of UTCs could be improved by altering cell culture conditions, cultures of umbilical-derived cells were grown in low oxygen conditions. Cells were seeded at 5000 cells/cm² in Growth Medium on gelatin coated flasks. Cells were initially cultured under standard atmospheric conditions through passage 5, at which point they were transferred to low oxygen (5% O₂) culture conditions.

Other growth conditions. In other protocols, cells were expanded on non-coated, collagen-coated, fibronectin-coated, laminin-coated and extracellular matrix protein-coated plates. Cultures have been demonstrated to expand well on these different matrices.

Results

Comparison of expansion potential of UTCs with other stem cell and non-stem cell populations. Both umbilical-derived and placenta-derived cells expanded for greater than 40 passages generating cell yields of >1E17 cells in 60 days. In contrast, MSCs and fibroblasts senesced after <25 days and <60 days, respectively. Although adipose-derived cells expanded for almost 60 days, they generated total cell yields of 4.5E12. Thus, when seeded at 5000 cells/cm² under the experimental conditions utilized, UTCs expanded much better than the other cell types grown under the same conditions (Table 2-1).

TABLE 2-1 Growth characteristics for different cell populations grown to senescence Total Population Total Cell Cell Type Senescence Doublings Yield MSC 24 d 8 4.72E7 Adipose 57 d 24 4.5E12 Fibroblasts 53 d 26 2.82E13 Umbilicus 65 d 42 6.15E17 Placenta 80 d 46 2.49E19

Expansion potential of cell banks at low density. Umbilicus-derived, placenta-derived and fibroblast cells expanded for greater than 10 passages generating cell yields of >1E11 cells in 60 days (Table 2-2). After 60 days under these conditions the fibroblasts became senescent whereas the umbilicus-derived and placenta-derived cell populations senesced after 80 days, completing >50 and >40 population doublings respectively.

TABLE 2-2 Growth characteristics for different cell populations using low density growth expansion from passage 10 till senescence Total Population Total Cell Cell Type Senescence Doublings Yield Fibroblast (P10) 80 d 43.68 2.59E11 Umbilicus (P10) 80 d 53.6 1.25E14 Placenta (P10) 60 d 32.96 6.09E12

Expansion of UTCs at low density from initial cell seeding. UTCs were expanded at low density (1,000 cells/cm²) on gelatin-coated and uncoated plates or flasks. Growth potential of these cells under these conditions was good. The cells expanded readily in a log phase growth. The rate of cell expansion was similar to that observed when placenta-derived cells were seeded at 5000 cells/cm² on gelatin-coated flasks in Growth Medium. No differences were observed in cell expansion potential between culturing on either uncoated flasks or gelatin-coated flasks. However, cells appeared phenotypically much smaller on gelatin-coated flasks and more larger cell phenotypes were observed on uncoated flasks.

Expansion of clonal neonatal or maternal placenta-derived cells. A clonal neonatal or maternal cell population can be expanded from placenta-derived cells isolated from the neonatal aspect or the maternal aspect, respectively, of the placenta. Cells are serially diluted and then seeded onto gelatin-coated plates in Growth medium for expansion at 1 cell/well in 96-well gelatin coated plates. From this initial cloning, expansive clones are identified, trypsinized, and reseeded in 12-well gelatin-coated plates in Growth medium and then subsequently passaged into T25 gelatin-coated flasks at 5,000 cells/cm² in Growth medium. Subcloning is performed to ensure that a clonal population of cells has been identified. For subcloning experiments, cells are trypsinized and reseeded at 0.5 cells/well. The subclones that grow well are expanded in gelatin-coated T25 flasks at 5,000 cells cm²/flask. Cells are passaged at 5,000 cells cm²/T75 flask. The growth characteristics of a clone may be plotted to demonstrate cell expansion. Karyotyping analysis can confirm that the clone is either neonatal or maternal.

Expansion of cells in low oxygen culture conditions. Cells expanded well under the reduced oxygen conditions, however, culturing under low oxygen conditions did not appear to have a significant effect on cell expansion of UTCs under the conditions used.

Summary. Cell expansion conditions comprising growing isolated umbilical cord tissue-derived cells at densities of about 5000 cells/cm², in Growth Medium on gelatin-coated or uncoated flasks, under standard atmospheric oxygen, are sufficient to generate large numbers of cells at passage 11. Furthermore, the data suggests that the cells can be readily expanded using lower density culture conditions (e.g. 1000 cells/cm²). umbilical cord tissue-derived cell expansion in low oxygen conditions also facilitates cell expansion, although no incremental improvement in cell expansion potential has yet been observed when utilizing these conditions for growth. Presently, culturing umbilical cord tissue-derived cells under standard atmospheric conditions is preferred for generating large pools of cells. However, when the culture conditions are altered, umbilical cord tissue-derived cell expansion can likewise be altered. This strategy may be used to enhance the proliferative and differentiative capacity of these cell populations.

Under the conditions utilized, while the expansion potential of MSC and adipose-derived cells is limited, umbilical cord tissue-derived cells expand readily to large numbers.

Example 3 Evaluation of Growth Media for Placenta-Derived Cells

Several cell culture media were evaluated for their ability to support the growth of placenta-derived cells. The growth of placenta-derived cells in normal (20%) and low (5%) oxygen was assessed after 3 days using the MTS colorimetric assay.

Methods & Materials

Placenta-derived cells at passage 8 (P8) were seeded at 1×10³ cells/well in 96 well plates in Growth Medium with penicillin/streptomycin. After 8 hours the medium was changed as described below and cells were incubated in normal (atmospheric) or low (5%, v/v) oxygen at 37° C., 5% CO₂ for 48 hours. MTS was added to the culture medium (Cell Titer 96® AQueous One Solution Cell Proliferation Assay, Promega, Madison, Wis.) for 3 hours and the absorbance measured at 490 nanometers (Molecular Devices, Sunnyvale Calif.).

TABLE 3-1 Culture Media Added fetal bovine serum % Culture Medium Supplier (v/v) DMEM low glucose Gibco Carlsbad CA 0, 2 10 DMEM high glucose Gibco 0, 2 10 RPMI 1640 Mediatech, Inc. 0, 2 10 Herndon, VA Cell gro-free (Serum-free, Mediatech, Inc. — Protein-free Ham's F10 Mediatech, Inc. 0, 2 10 MSCGM (complete with serum) Cambrex, 0, 2 10 Walkersville, MD Complete-serum free w/albumin Mediatech, Inc. — Growth Medium NA — Ham's F12 Mediatech, Inc. 0, 2 10 Iscove's Mediatech, Inc. 0, 2 10 Basal Medium Eagle's Mediatech, Inc. DMEM/F12 (1:1) Mediatech, Inc. 0, 2 10

Results

Standard curves for the MTS assay established a linear correlation between an increase in absorbance and an increase in cell number. The absorbance values obtained were converted into estimated cell numbers and the change (%) relative to the initial seeding was calculated.

The Effect of Serum. The addition of serum to media at normal oxygen conditions resulted in a reproducible dose-dependent increase in absorbance and thus the viable cell number. The addition of serum to complete MSCGM resulted in a dose-dependent decrease in absorbance. In the media without added serum, cells only grew appreciably in Cellgro FREE™, Ham's F10 and DMEM.

The Effect of Oxygen. Reduced oxygen appeared to increase the growth rate of cells in Growth Medium, Ham's F10, and MSCGM. In decreasing order of growth, the media resulting in the best growth of the cells were Growth Medium>MSCGM>Iscove's+10% FBS=DMEM-H+10% FBS=Ham's F12+10% FBS=RPMI 1640+10% FBS.

Summary. Placenta-derived cells may be grown in a variety of culture media in normal or low oxygen. Short term growth of placenta-derived cells was determined in twelve basal media with 0, 2 and 10% (v/v) serum in 5% or atmospheric oxygen. In general, placenta-derived cells did not grow as well in serum-free conditions with the exception of Ham's F10 and Cellgro FREE™, which are also protein-free. Growth in these serum-free media was about 25-33% of the maximal growth observed with media containing 15% serum.

Example 4 Growth of Umbilical Cord Tissue and Placenta-Derived Cells in Medium Containing D-Valine

It has been reported that medium containing D-valine instead of the normal L-valine isoform can be used to selectively inhibit the growth of fibroblast-like cells in culture (Hongpaisan J. 2000. Cell Biol Int. 24:1-7; Sordillo et al. 1988. Cell Biol Int Rep. 12:355-64). It was not previously known whether umbilical cord tissue and placenta-derived cells could grow in medium containing D-valine.

Methods & Materials

Placenta-derived cells (P3), fibroblasts (P9) and umbilical-derived cells (P5) were seeded at 5×10³ cells/cm² in gelatin-coated T75 flasks (Corning, Corning, N.Y.). After 24 hours the medium was removed and the cells were washed with phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif.) to remove residual medium. The medium was replaced with a Modified Growth Medium (DMEM with D-valine (special order Gibco), 15% (v/v) dialyzed fetal bovine serum (Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma), penicillin/streptomycin (Gibco)).

Results

Placenta-derived, umbilical-derived, and fibroblast cells seeded in the D-valine-containing medium did not proliferate, unlike cells seeded in Growth Medium containing dialyzed serum. Fibroblasts cells changed morphologically, increasing in size and changing shape. All of the cells died and eventually detached from the flask surface after 4 weeks. These results indicate that medium containing D-valine is not suitable for selectively growing umbilical-derived cells.

Example 5 Cryopreservation Media for Placenta-Derived Cells

Cryopreservation media for the cryopreservation of placenta-derived cells were evaluated.

Methods & Materials

Placenta-derived cells grown in Growth Medium in a gelatin-coated T75 flask were washed with PBS and trypsinized using 1 milliliter Trypsin/EDTA (Gibco). The trypsinization was stopped by adding 10 milliliters Growth Medium. The cells were centrifuged at 150×g, supernatant removed, and the cell pellet was resuspended in 1 milliliter Growth Medium. An aliquot of cell suspension, 60 microliters, was removed and added to 60 microliters trypan blue (Sigma). The viable cell number was estimated using a hemocytometer. The cell suspension was divided into four equal aliquots each containing 88×10⁴ cells each. The cell suspension was centrifuged and resuspended in 1 milliliter of each media below and transferred into Cryovials (Nalgene).

-   -   1.) Growth Medium+10% (v/v) DMSO (Hybrimax, Sigma, St. Louis,         Mo.)     -   2.) Cell Freezing medium w/DMSO, w/methyl cellulose, serum-free         (C6295, Sigma, St. Louis, Mo.)     -   3.) Cell Freezing medium serum-free (C2639, Sigma, St. Louis,         Mo.)     -   4.) Cell Freezing Medium w/glycerol (C6039, Sigma, St. Louis,         Mo.)         The cells were cooled at approximately −1° C./min overnight in a         −80° C. freezer using a “Mr Frosty” freezing container according         to the manufacturer's instructions (Nalgene, Rochester, N.Y.).         Vials of cells were transferred into liquid nitrogen for 2 days         before thawing rapidly in a 37° C. water bath. The cells were         added to 10 milliliters Growth Medium and centrifuged before the         cell number and viability was estimated. Cells were seeded onto         gelatin-coated flasks at 5,000 cells/cm² to determine whether         the cells would attach and proliferate.

Results

The initial viability of the cells to be cryopreserved was assessed by trypan blue staining to be 100%. The initial viability of the cells to be cryopreserved was assessed by trypan blue staining to be 100%.

There was a commensurate reduction in cell number with viability for C6295 due to cells lysis. The viable cells cryopreserved in all four solutions attached, divided, and produced a confluent monolayer within 3 days. There was no discernable difference in estimated growth rate.

Summary. The cryopreservation of cells is one procedure available for preparation of a cell bank or a cell product. Four cryopreservation mixtures were compared for their ability to protect human placenta-derived cells from freezing damage. Dulbecco's modified Eagle's medium (DMEM) and 10% (v/v) dimethylsulfoxide (DMSO) is the preferred medium of those compared for cryopreservation of placenta-derived cells.

Example 6 Karyotype Analysis of Umbilical Cord Tissue and Placenta-Derived Cells

Cell lines used in cell therapy are preferably homogeneous and free from any contaminating cell type. Cells used in cell therapy should have a normal chromosome number (46) and structure. To identify placenta- and umbilicus-derived cell lines that are homogeneous and free from cells of non-postpartum tissue origin, karyotypes of cell samples were analyzed.

Materials and Methods

UTCs from postpartum tissue of a male neonate were cultured in Growth Medium containing penicillin/streptomycin. Postpartum tissue from a male neonate (X,Y) was selected to allow distinction between neonatal-derived cells and maternal derived cells (X,X). Cells were seeded at 5,000 cells per square centimeter in Growth Medium in a T25 flask (Corning, Corning, N.Y.) and expanded to 80% confluence. A T25 flask containing cells was filled to the neck with Growth Medium. Samples were delivered to a clinical cytogenetics laboratory by courier (estimated lab to lab transport time is one hour). Cells were analyzed during metaphase when the chromosomes are best visualized. Of twenty cells in metaphase counted, five were analyzed for normal homogeneous karyotype number (two). A cell sample was characterized as homogeneous if two karyotypes were observed. A cell sample was characterized as heterogeneous if more than two karyotypes were observed. Additional metaphase cells were counted and analyzed when a heterogeneous karyotype number (four) was identified.

Results

All cell samples sent for chromosome analysis were interpreted as exhibiting a normal appearance. Three of the sixteen cell lines analyzed exhibited a heterogeneous phenotype (XX and XY) indicating the presence of cells derived from both neonatal and maternal origins (Table 6-1). Cells derived from tissue Placenta-N were isolated from the neonatal aspect of placenta. At passage zero, this cell line appeared homogeneous XY. However, at passage nine, the cell line was heterogeneous (XX/XY), indicating a previously undetected presence of cells of maternal origin.

TABLE 6-1 Results of cell karyotype analysis Metaphase Metaphase cells cells Number of ISCN Tissue passage counted analyzed karyotype Karyotype Placenta 22 20 5 2 46, XX Umbilical 23 20 5 2 46, XX Umbilical 6 20 5 2 46, XY Placenta 2 20 5 2 46, XX Umbilical 3 20 5 2 46, XX Placenta-N 0 20 5 2 46, XY Placenta-V 0 20 5 2 46, XY Placenta-M 0 21 5 4 46, XY[18]/ 46, XX[3] Placenta-M 4 20 5 2 46, XX Placenta-N 9 25 5 4 46, XY[5]/ 46, XX[20] Placenta-N 1 20 5 2 46, XY C1 Placenta-N 1 20 6 4 46, XY[2]/ C3 46, XX[18] Placenta-N 1 20 5 2 46, XY C4 Placenta-N 1 20 5 2 46, XY C15 Placenta-N 1 20 5 2 46, XY C20 Key: N—Neonatal aspect; V—villous region; M—maternal aspect; C—clone

Summary. Chromosome analysis identified placenta- and umbilicus-derived cells whose karyotypes appeared normal as interpreted by a clinical cytogenetic laboratory. Karyotype analysis also identified cell lines free from maternal cells, as determined by homogeneous karyotype.

Example 7 Evaluation of Human Umbilical Cord Tissue and Placenta-Derived Cell Surface Markers by Flow Cytometry

Characterization of cell surface proteins or “markers” by flow cytometry can be used to determine a cell line's identity. The consistency of expression can be determined from multiple donors, and in cells exposed to different processing and culturing conditions. Cell lines isolated from the placenta and umbilicus were characterized (by flow cytometry), providing a profile for the identification of these cell lines.

Materials and Methods

Media and culture vessels. Cells were cultured in Growth Medium (Gibco Carlsbad, Calif.) with penicillin/streptomycin. Cells were cultured in plasma-treated T75, T150, and T225 tissue culture flasks (Corning, Corning, N.Y.) until confluent. The growth surfaces of the flasks were coated with gelatin by incubating 2% (w/v) gelatin (Sigma, St. Louis, Mo.) for 20 minutes at room temperature.

Antibody Staining and flow cytometry analysis. Adherent cells in flasks were washed in PBS and detached with Trypsin/EDTA. Cells were harvested, centrifuged, and resuspended in 3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter. In accordance to the manufacture's specifications, antibody to the cell surface marker of interest (see below) was added to one hundred microliters of cell suspension and the mixture was incubated in the dark for 30 minutes at 4° C. After incubation, cells were washed with PBS and centrifuged to remove unbound antibody. Cells were resuspended in 500 microliter PBS and analyzed by flow cytometry. Flow cytometry analysis was performed with a FACScalibur instrument (Becton Dickinson, San Jose, Calif.).

The following antibodies to cell surface markers were used.

Catalog Antibody Manufacture Number CD10 BD Pharmingen (San Diego, CA) 555375 CD13 BD Pharmingen 555394 CD31 BD Pharmingen 555446 CD34 BD Pharmingen 555821 CD44 BD Pharmingen 555478 CD45RA BD Pharmingen 555489 CD73 BD Pharmingen 550257 CD90 BD Pharmingen 555596 CD117 BD Pharmingen 340529 CD141 BD Pharmingen 559781 PDGFr-alpha BD Pharmingen 556002 HLA-A, B, C BD Pharmingen 555553 HLA-DR, DP, DQ BD Pharmingen 555558 IgG-FITC Sigma (St. Louis, MO) F-6522 IgG-PE Sigma P-4685

Placenta and umbilicus comparison. Placenta-derived cells were compared to umbilicus-derived cells at passage 8.

Passage to passage comparison. Placenta- and umbilicus-derived cells were analyzed at passages 8, 15, and 20.

Donor to donor comparison. To compare differences among donors, placenta-derived cells from different donors were compared to each other, and umbilicus-derived cells from different donors were compared to each other.

Surface coating comparison. Placenta-derived cells cultured on gelatin-coated flasks was compared to placenta-derived cells cultured on uncoated flasks. Umbilicus-derived cells cultured on gelatin-coated flasks was compared to umbilicus-derived cells cultured on uncoated flasks.

Digestion enzyme comparison. Four treatments used for isolation and preparation of cells were compared. Cells isolated from placenta by treatment with 1) collagenase; 2) collagenase/dispase; 3) collagenase/hyaluronidase; and 4) collagenase/hyaluronidase/dispase were compared.

Placental layer comparison. Cells derived from the maternal aspect of placental tissue were compared to cells derived from the villous region of placental tissue and cells derived from the neonatal fetal aspect of placenta.

Results

Placenta vs. umbilicus comparison. Placenta- and umbilicus-derived cells analyzed by flow cytometry showed positive expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, indicated by the increased values of fluorescence relative to the IgG control. These cells were negative for detectable expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated by fluorescence values comparable to the IgG control. Variations in fluorescence values of positive curves were accounted for. The mean (i.e. CD13) and range (i.e. CD90) of the positive curves showed some variation, but the curves appeared normal, confirming a homogenous population. Both curves individually exhibited values greater than the IgG control.

Passage to passage comparison—placenta-derived cells. Placenta-derived cells at passages 8, 15, and 20 analyzed by flow cytometry all were positive for expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, as reflected in the increased value of fluorescence relative to the IgG control. The cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ having fluorescence values consistent with the IgG control.

Passage to passage comparison—umbilicus-derived cells. Umbilicus-derived cells at passage 8, 15, and 20 analyzed by flow cytometry all expressed CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, indicated by increased fluorescence relative to the IgG control. These cells were negative for CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated by fluorescence values consistent with the IgG control.

Donor to donor comparison—placenta-derived cells. Placenta-derived cells isolated from separate donors analyzed by flow cytometry each expressed CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, with increased values of fluorescence relative to the IgG control. The cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence value consistent with the IgG control.

Donor to donor comparison—umbilicus derived cells. Umbilicus-derived cells isolated from separate donors analyzed by flow cytometry each showed positive expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values of fluorescence relative to the IgG control. These cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ with fluorescence values consistent with the IgG control.

The effect of surface coating with gelatin on placenta-derived cells. Placenta-derived cells expanded on either gelatin-coated or uncoated flasks analyzed by flow cytometry all expressed of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values of fluorescence relative to the IgG control. These cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ indicated by fluorescence values consistent with the IgG control.

The effect of surface coating with gelatin on umbilicus-derived cells. Umbilicus-derived cells expanded on gelatin and uncoated flasks analyzed by flow cytometry all were positive for expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, with increased values of fluorescence relative to the IgG control. These cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, with fluorescence values consistent with the IgG control.

Effect of enzyme digestion procedure used for preparation of the cells on the cell surface marker profile. Placenta-derived cells isolated using various digestion enzymes analyzed by flow cytometry all expressed CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, as indicated by the increased values of fluorescence relative to the IgG control. These cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence values consistent with the IgG control.

Placental layer comparison. Cells isolated from the maternal, villous, and neonatal layers of the placenta, respectively, analyzed by flow cytometry showed positive expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, as indicated by the increased value of fluorescence relative to the IgG control. These cells were negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence values consistent with the IgG control.

Summary. Analysis of placenta- and umbilicus-derived cells by flow cytometry has established of an identity of these cell lines. Placenta- and umbilicus-derived cells are positive for CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A,B,C and negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, DP, DQ. This identity was consistent between variations in variables including the donor, passage, culture vessel surface coating, digestion enzymes, and placental layer. Some variation in individual fluorescence value histogram curve means and ranges was observed, but all positive curves under all conditions tested were normal and expressed fluorescence values greater than the IgG control, thus confirming that the cells comprise a homogenous population that has positive expression of the markers.

Example 8 Immunohistochemical Characterization of Umbilical Cord and Placental Tissue Phenotypes

The phenotypes of cells found within human umbilical cord and placenta were analyzed by immunohistochemistry.

Materials & Methods

Tissue Preparation. Human umbilical cord and placenta tissue was harvested and immersion fixed in 4% (w/v) paraformaldehyde overnight at 4° C. Immunohistochemistry was performed using antibodies directed against the following epitopes:vimentin (1:500; Sigma, St. Louis, Mo.), desmin (1:150, raised against rabbit; Sigma; or 1:300, raised against mouse; Chemicon, Temecula, Calif.), alpha-smooth muscle actin (SMA; 1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF; 1:200; Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation, Carpinteria, Calif.). In addition, the following markers were tested: anti-human GROalpha-PE (1:100; Becton Dickinson, Franklin Lakes, N.J.), anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.), anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa Cruz Biotech), and anti-human NOGO-A (1:100; Santa Cruz Biotech). Fixed specimens were trimmed with a scalpel and placed within OCT embedding compound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on a dry ice bath containing ethanol. Frozen blocks were then sectioned (10 μm thick) using a standard cryostat (Leica Microsystems) and mounted onto glass slides for staining.

Immunohistochemistry. Immunohistochemistry was performed similar to previous studies (e.g., Messina, et al., 2003, Exper. Neurol. 184: 816-829). Tissue sections were washed with phosphate-buffered saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 1 hour to access intracellular antigens. In instances where the epitope of interest would be located on the cell surface (CD34, ox-LDL R1), Triton was omitted in all steps of the procedure in order to prevent epitope loss. Furthermore, in instances where the primary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v) donkey serum was used in place of goat serum throughout the procedure. Primary antibodies, diluted in blocking solution, were then applied to the sections for a period of 4 hours at room temperature. Primary antibody solutions were removed, and cultures washed with PBS prior to application of secondary antibody solutions (1 hour at room temperature) containing block along with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbit IgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG—FITC (1:150; Santa Cruz Biotech). Cultures were washed, and 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using the appropriate fluorescence filter on an Olympus inverted epi-fluorescent microscope (Olympus, Melville, N.Y.). Positive staining was represented by fluorescence signal above control staining Representative images were captured using a digital color video camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, each image was taken using only one emission filter at a time. Layered montages were then prepared using Adobe Photoshop software (Adobe, San Jose, Calif.).

Results

Umbilical cord characterization. Vimentin, desmin, SMA, CK18, vWF, and CD34 markers were expressed in a subset of the cells found within umbilical cord. In particular, vWF and CD34 expression were restricted to blood vessels contained within the cord. CD34+ cells were on the innermost layer (lumen side). Vimentin expression was found throughout the matrix and blood vessels of the cord. SMA was limited to the matrix and outer walls of the artery and vein, but not contained with the vessels themselves. CK18 and desmin were observed within the vessels only, desmin being restricted to the middle and outer layers.

Placenta characterization. Vimentin, desmin, SMA, CK18, vWF, and CD34 were all observed within the placenta and regionally specific.

GROalpha, GCP-2, ox-LDL R1, and NOGO-A Tissue Expression. None of these markers were observed within umbilical cord or placental tissue.

Summary. Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18, von Willebrand Factor, and CD 34 are expressed in cells within human umbilical cord and placenta.

Example 9 Analysis of Cells using Oligonucleotide Arrays

Affymetrix GENECHIP® arrays were used to compare gene expression profiles of umbilicus- and placenta-derived cells with fibroblasts, human mesenchymal stem cells, and another cell line derived from human bone marrow. This analysis provided a characterization of the cells and identified unique molecular markers for these cells.

Materials and Methods

Isolation and culture of cells. Human umbilical cords and placenta were obtained from National Disease Research Interchange (NDRI, Philadelphia, Pa.) from normal full term deliveries with patient consent. The tissues were received and cells were isolated as described in Example 1. Cells were cultured in Growth Medium (using DMEM-LG) on gelatin-coated tissue culture plastic flasks. The cultures were incubated at 37° C. with 5% CO₂.

Human dermal fibroblasts were purchased from Cambrex Incorporated (Walkersville, Md.; Lot number 9F0844) and ATCC CRL-1501 (CCD39SK). Both lines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, Calif.) with 10% (v/v) fetal bovine serum (Hyclone) and penicillin/streptomycin (Invitrogen). The cells were grown on standard tissue-treated plastic.

Human mesenchymal stem cells (hMSC) were purchased from Cambrex Incorporated (Walkersville, Md.; Lot numbers 2F1655, 2F1656 and 2F1657) and cultured according to the manufacturer's specifications in MSCGM Media (Cambrex). The cells were grown on standard tissue cultured plastic at 37° C. with 5% CO₂.

Human iliac crest bone marrow was received from NDRI with patient consent. The marrow was processed according to the method outlined by Ho, et al. (WO03/025149). The marrow was mixed with lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA, pH 7.2) at a ratio of 1 part bone marrow to 20 parts lysis buffer. The cell suspension was vortexed, incubated for 2 minutes at ambient temperature, and centrifuged for 10 minutes at 500×g. The supernatant was discarded and the cell pellet was resuspended in Minimal Essential Medium-alpha (Invitrogen) supplemented with 10% (v/v) fetal bovine serum and 4 mM glutamine. The cells were centrifuged again and the cell pellet was resuspended in fresh medium. The viable mononuclear cells were counted using trypan-blue exclusion (Sigma, St. Louis, Mo.). The mononuclear cells were seeded in tissue-cultured plastic flasks at 5×10⁴ cells/cm². The cells were incubated at 37° C. with 5% CO₂ at either standard atmospheric O₂ or at 5% O₂. Cells were cultured for 5 days without a media change. Media and non-adherent cells were removed after 5 days of culture. The adherent cells were maintained in culture.

Isolation of mRNA and GENECHIP® Analysis. Actively growing cultures of cells were removed from the flasks with a cell scraper in cold PBS. The cells were centrifuged for 5 minutes at 300×g. The supernatant was removed and the cells were resuspended in fresh PBS and centrifuged again. The supernatant was removed and the cell pellet was immediately frozen and stored at −80° C. Cellular mRNA was extracted and transcribed into cDNA, which was then transcribed into cRNA and biotin-labeled. The biotin-labeled cRNA was hybridized with HG-U133A GENECHIP® oligonucleotide array (Affymetrix, Santa Clara Calif.). The hybridization and data collection was performed according to the manufacturer's specifications. Analyses were performed using “Significance Analysis of Microarrays” (SAM) version 1.21 computer software (Stanford University; Tusher, V. G. et al., 2001, Proc. Natl. Acad. Sci. USA 98: 5116-5121).

Results

Fourteen different populations of cells were analyzed. The cells along with passage information, culture substrate, and culture media are listed in Table 9-1.

TABLE 9-1 Cells analyzed by the microarray study. Pas- Cell Population sage Substrate Medium Umbilicus (022803) 2 Gelatin DMEM, 15% FBS, 2-ME Umbilicus (042103) 3 Gelatin DMEM, 15% FBS, 2-ME Umbilicus (071003) 4 Gelatin DMEM, 15% FBS, 2-ME Placenta (042203) 12 Gelatin DMEM, 15% FBS, 2-ME Placenta (042903) 4 Gelatin DMEM, 15% FBS, 2-ME Placenta (071003) 3 Gelatin DMEM, 15% FBS, 2-ME ICBM (070203) (5% O2) 3 Plastic MEM, 10% FBS ICBM (062703) (std. O2) 5 Plastic MEM, 10% FBS ICBM (062703) (5% O2) 5 Plastic MEM, 10% FBS hMSC (Lot 2F1655) 3 Plastic MSCGM hMSC (Lot 2F1656) 3 Plastic MSCGM hMSC (Lot 2F 1657) 3 Plastic MSCGM hFibroblast (9F0844) 9 Plastic DMEM-F12, 10% FBS hFibroblast (CCD39SK) 4 Plastic DMEM-F12, 10% FBS Cell lines are listed by identification code along with passage at time of analysis, cell growth substrate and growth medium.

The data were evaluated by a Principle Component Analysis, analyzing the 290 genes that were differentially expressed in the cells. This analysis allows for a relative comparison for the similarities between the populations. Table 9-2 shows the Euclidean distances that were calculated for the comparison of the cell pairs. The Euclidean distances were based on the comparison of the cells based on the 290 genes that were differentially expressed among the cell types. The Euclidean distance is inversely proportional to similarity between the expression of the 290 genes (i.e., the greater the distance, the less similarity exists).

TABLE 9-2 The Euclidean Distances for the Cell Pairs. Cell Pair Euclidean Distance ICBM-hMSC 24.71 Placenta-umbilical 25.52 ICBM-Fibroblast 36.44 ICBM-placenta 37.09 Fibroblast-MSC 39.63 ICBM-Umbilical 40.15 Fibroblast-Umbilical 41.59 MSC-Placenta 42.84 MSC-Umbilical 46.86 ICBM-placenta 48.41

Tables 9-3, 9-4, and 9-5 show the expression of genes increased in placenta-derived cells (Table 9-3), increased in umbilicus-derived cells (Table 9-4), and reduced in umbilicus- and placenta-derived cells (Table 9-5). The column entitled “Probe Set ID” refers to the manufacturer's identification code for the sets of several oligonucleotide probes located on a particular site on the chip, which hybridize to the named gene (column “Gene Name”), comprising a sequence that can be found within the NCBI (GenBank) database at the specified accession number (column “NCBI Accession Number”).

TABLE 9-3 Genes shown to have specifically increased expression in the placenta- derived cells as compared to other cell lines assayed NCBI Probe Accession Set ID Gene Name Number 209732_at C-type (calcium dependent, carbohydrate- AF070642 recognition domain) lectin, superfamily member 2 (activation-induced) 206067_s_at Wilms tumor 1 NM_024426 207016_s_at aldehyde dehydrogenase 1 family, AB015228 member A2 206367_at renin NM_000537 210004_at oxidized low density lipoprotein (lectin- AF035776 like) receptor 1 214993_at Homo sapiens, clone IMAGE: 4179671, AF070642 mRNA, partial cds 202178_at protein kinase C, zeta NM_002744 209780_at hypothetical protein DKFZp564F013 AL136883 204135_at downregulated in ovarian cancer 1 NM_014890 213542_at Homo sapiens mRNA; cDNA AI246730 DKFZp547K1113 (from clone DKFZp547K1113)

TABLE 9-4 Genes shown to have specifically increased expression in the umbilicus- derived cells as compared to other cell lines assayed NCBI Probe Set Accession ID Gene Name Number 202859_x_at interleukin 8 NM_000584 211506_s_at interleukin 8 AF043337 210222_s_at reticulon 1 BC000314 204470_at chemokine (C-X-C motif) ligand 1 NM_001511 (melanoma growth stimulating activity 206336_at chemokine (C-X-C motif) ligand 6 NM_002993 (granulocyte chemotactic protein 2) 207850_at chemokine (C-X-C motif) ligand 3 NM_002090 203485_at reticulon 1 NM_021136 202644_s_at tumor necrosis factor, alpha-induced NM_006290 protein 3

TABLE 9-5 Genes shown to have decreased expression in umbilicus- and placenta- derived cells as compared to other cell lines assayed NCBI Probe Set Accession ID Gene name Number 210135_s_at short stature homeobox 2 AF022654.1 205824_at heat shock 27 kDa protein 2 NM_001541.1 209687_at chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) U19495.1 203666_at chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) NM_000609.1 212670_at elastin (supravalvular aortic stenosis, Williams-Beuren syndrome) AA479278 213381_at Homo sapiens mRNA; cDNA DKFZp586M2022 (from clone N91149 DKFZp586M2022) 206201_s_at mesenchyme home obox 2 (growth arrest-specific homeo box) NM_005924.1 205817_at sine oculis homeobox homolog 1 (Drosophila) NM_005982.1 209283_at crystallin, alpha B AF007162.1 212793_at dishevelled associated activator of morphogenesis 2 BF513244 213488_at DKFZP586B2420 protein AL050143.1 209763_at similar to neuralin 1 AL049176 205200_at tetranectin (plasminogen binding protein) NM_003278.1 205743_at src homology three (SH3) and cysteine rich domain NM_003149.1 200921_s_at B-cell translocation gene 1, anti-proliferative NM_001731.1 206932_at cholesterol 25-hydroxylase NM_003956.1 204198_s_at runt-related transcription factor 3 AA541630 219747_at hypothetical protein FLJ23191 NM_024574.1 204773_at interleukin 11 receptor, alpha NM_004512.1 202465_at procollagen C-endopeptidase enhancer NM_002593.2 203706_s_at frizzled homolog 7 (Drosophila) NM_003507.1 212736_at hypothetical gene BC008967 BE299456 214587_at collagen, type VIII, alpha 1 BE877796 201645_at tenascin C (hexabrachion) NM_002160.1 210239_at iroquois homeobox protein 5 U90304.1 203903_s_at hephaestin NM_014799.1 205816_at integrin, beta 8 NM_002214.1 203069_at synaptic vesicle glycoprotein 2 NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis, clone MAMMA1001744 AU147799 206315_at cytokine receptor-like factor 1 NM_004750.1 204401_at potassium intermediate/small conductance calcium-activated channel, NM_002250.1 subfamily N, member 4 216331_at integrin, alpha 7 AK022548.1 209663_s_at integrin, alpha 7 AF072132.1 213125_at DKFZP586L151 protein AW007573 202133_at transcriptional co-activator with PDZ-binding motif (TAZ) AA081084 206511_s_at sine oculis homeobox homolog 2 (Drosophila) NM_016932.1 213435_at KIAA1034 protein AB028957.1 206115_at early growth response 3 NM_004430.1 213707_s_at distal-less homeo box 5 NM_005221.3 218181_s_at hypothetical protein FLJ20373 NM_017792.1 209160_at aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid AB018580.1 dehydrogenase, type II) 213905_x_at biglycan AA845258 201261_x_at biglycan BC002416.1 202132_at transcriptional co-activator with PDZ-binding motif (TAZ) AA081084 214701_s_at fibronectin 1 AJ276395.1 213791_at proenkephalin NM_006211.1 205422_s_at integrin, beta-like 1 (with EGF-like repeat domains) NM_004791.1 214927_at Homo sapiens mRNA full length insert cDNA clone EUROIMAGE AL359052.1 1968422 206070_s_at EphA3 AF213459.1 212805_at KIAA0367 protein AB002365.1 219789_at natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic AI628360 peptide receptor C) 219054_at hypothetical protein FLJ14054 NM_024563.1 213429_at Homo sapiens mRNA; cDNA DKFZp564B222 (from clone AW025579 DKFZp564B222) 204929_s_at vesicle-associated membrane protein 5 (myobrevin) NM_006634.1 201843_s_at EGF-containing fibulin-like extracellular matrix protein 1 NM_004105.2 221478_at BCL2/adenovirus E1B 19 kDa interacting protein 3-like AL132665.1 201792_at AE binding protein 1 NM_001129.2 204570_at cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) NM_001864.1 201621_at neuroblastoma, suppression of tumorigenicity 1 NM_005380.1 202718_at insulin-like growth factor binding protein 2, 36 kDa NM_000597.1

Tables 9-6, 9-7, and 9-8 show the expression of genes increased in human fibroblasts (Table 9-6), ICBM cells (Table 9-7), and MSCs (Table 9-8).

TABLE 9-6 Genes that were shown to have increased expression in fibroblasts as compared to the other cell lines assayed. dual specificity phosphatase 2 KIAA0527 protein Homo sapiens cDNA: FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic, intermediate polypeptide 1 ankyrin 3, node of Ranvier (ankyrin G) inhibin, beta A (activin A, activin AB alpha polypeptide) ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053 protein microtubule-associated protein 1A zinc finger protein 41 HSPC019 protein Homo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNA DKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to rat protein kinase C-binding enigma) inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein hypothetical protein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderately similar to cytokine receptor-like factor 2; cytokine receptor CRL2 precursor [Homo sapiens] transforming growth factor, beta 2 hypothetical protein MGC29643 antigen identified by monoclonal antibody MRC OX-2 putative X-linked retinopathy protein

TABLE 9-7 Genes that were shown to have increased expression in the ICBM- derived cells as compared to the other cell lines assayed cardiac ankyrin repeat protein MHC class I region ORF integrin, alpha 10 hypothetical protein FLJ22362 UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetyl- galactosaminyltransferase 3 (GalNAc-T3) interferon-induced protein 44 SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal) keratin associated protein 1-1 hippocalcin-like 1 jagged 1 (Alagille syndrome) proteoglycan 1, secretory granule

TABLE 9-8 Genes that were shown to have increased expression in the MSC cells as compared to the other cell lines assayed. interleukin 26 maltase-glucoamylase (alpha-glucosidase) nuclear receptor subfamily 4, group A, member 2 v-fos FBJ murine osteosarcoma viral oncogene homolog hypothetical protein DC42 nuclear receptor subfamily 4, group A, member 2 FBJ murine osteosarcoma viral oncogene homolog B WNT1 inducible signaling pathway protein 1 MCF.2 cell line derived transforming sequence potassium channel, subfamily K, member 15 cartilage paired-class homeoprotein 1 Homo sapiens cDNA FLJ12232 fis, clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, clone LIVER2000775 jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc finger protein 51) zinc finger protein 36, C3H type, homolog (mouse)

Summary. The present examination was performed to provide a molecular characterization of the cells derived from umbilical cord and placenta. This analysis included cells derived from three different umbilical cords and three different placentas. The examination also included two different lines of dermal fibroblasts, three lines of mesenchymal stem cells, and three lines of iliac crest bone marrow cells. The mRNA that was expressed by these cells was analyzed using an oligonucleotide array that contained probes for 22,000 genes. Results showed that 290 genes are differentially expressed in these five different cell types. These genes include ten genes that are specifically increased in the placenta-derived cells and seven genes specifically increased in the umbilical cord-derived cells. Fifty-four genes were found to have specifically lower expression levels in placenta and umbilical cord, as compared with the other cell types. The expression of selected genes has been confirmed by PCR (see the example that follows). These results demonstrate that the cells have a distinct gene expression profile, for example, as compared to bone marrow-derived cells and fibroblasts.

Example 10 Cell Markers in Umbilical Cord Tissue and Placental-Derived Cells

In the preceding example, similarities and differences in cells derived from the human placenta and the human umbilical cord were assessed by comparing their gene expression profiles with those of cells derived from other sources (using an oligonucleotide array). Six “signature” genes were identified: oxidized LDL receptor 1, interleukin-8, renin, reticulon, chemokine receptor ligand 3 (CXC ligand 3), and granulocyte chemotactic protein 2 (GCP-2). These “signature” genes were expressed at relatively high levels in the cells.

The procedures described in this example were conducted to verify the microarray data and find concordance/discordance between gene and protein expression, as well as to establish a series of reliable assay for detection of unique identifiers for placenta- and umbilicus-derived cells.

Methods & Materials

Cells. Placenta-derived cells (three isolates, including one isolate predominately neonatal as identified by karyotyping analysis), umbilicus-derived cells (four isolates), and Normal Human Dermal Fibroblasts (NHDF; neonatal and adult) grown in Growth Medium with penicillin/streptomycin in a gelatin-coated T75 flask. Mesechymal Stem Cells (MSCs) were grown in Mesenchymal Stem Cell Growth Medium Bullet kit (MSCGM; Cambrex, Walkerville, Md.).

For the IL-8 protocol, cells were thawed from liquid nitrogen and plated in gelatin-coated flasks at 5,000 cells/cm², grown for 48 hours in Growth Medium and then grown for further 8 hours in 10 milliliters of serum starvation medium (DMEM—low glucose (Gibco, Carlsbad, Calif.), penicillin/streptomycin (Gibco, Carlsbad, Calif.) and 0.1% (w/v) Bovine Serum Albumin (BSA; Sigma, St. Louis, Mo.)). After this treatment RNA was extracted and the supernatants were centrifuged at 150×g for 5 minutes to remove cellular debris. Supernatants were then frozen at −80° C. for ELISA analysis.

Cell culture for ELISA assay. Cells derived from placenta and umbilicus, as well as human fibroblasts derived from human neonatal foreskin were cultured in Growth Medium in gelatin-coated T75 flasks. Cells were frozen at passage 11 in liquid nitrogen. Cells were thawed and transferred to 15-milliliter centrifuge tubes. After centrifugation at 150×g for 5 minutes, the supernatant was discarded. Cells were resuspended in 4 milliliters culture medium and counted. Cells were grown in a 75 cm² flask containing 15 milliliters of Growth Medium at 375,000 cell/flask for 24 hours. The medium was changed to a serum starvation medium for 8 hours. Serum starvation medium was collected at the end of incubation, centrifuged at 14,000×g for 5 minutes (and stored at −20° C.).

To estimate the number of cells in each flask, 2 milliliters of tyrpsin/EDTA (Gibco, Carlsbad, Calif.) was added each flask. After cells detached from the flask, trypsin activity was neutralized with 8 milliliters of Growth Medium. Cells were transferred to a 15 milliliters centrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant was removed and 1 milliliter Growth Medium was added to each tube to resuspend the cells. Cell number was estimated using a hemocytometer.

ELISA assay. The amount of IL-8 secreted by the cells into serum starvation medium was analyzed using ELISA assays (R&D Systems, Minneapolis, Minn.). All assays were tested according to the instructions provided by the manufacturer.

Total RNA isolation. RNA was extracted from confluent umbilicus and placenta-derived cells and fibroblasts or for IL-8 expression from cells treated as described above. Cells were lysed with 350 microliters buffer RLT containing beta-mercaptoethanol (Sigma, St. Louis, Mo.) according to the manufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, Calif.). RNA was extracted according to the manufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, Calif.) and subjected to DNase treatment (2.7 U/sample) (Sigma St. Louis, Mo.). RNA was eluted with 50 microliters DEPC-treated water and stored at −80° C.

Reverse transcription. RNA was also extracted from human placenta and umbilicus. Tissue (30 milligram) was suspended in 700 microliters of buffer RLT containing 2-mercaptoethanol. Samples were mechanically homogenized and the RNA extraction proceeded according to manufacturer's specification. RNA was extracted with 50 microliters of DEPC-treated water and stored at −80° C. RNA was reversed transcribed using random hexamers with the TaqMan reverse transcription reagents (Applied Biosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60 minutes, and 95° C. for 10 minutes. Samples were stored at −20° C.

Genes identified by cDNA microarray as uniquely regulated in postpartum cells (signature genes—including oxidized LDL receptor, interleukin-8, renin and reticulon), were further investigated using real-time and conventional PCR.

Real-time PCR. PCR was performed on cDNA samples using Assays-on-Demand™ gene expression products: oxidized LDL receptor (Hs00234028); renin (Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2 (Hs00605742); IL-8 (Hs00174103); and GAPDH (Applied Biosystems, Foster City, Calif.) were mixed with cDNA and TaqMan Universal PCR master mix according to the manufacturer's instructions (Applied Biosystems, Foster City, Calif.) using a 7000 sequence detection system with ABI Prism 7000 SDS software (Applied Biosystems, Foster City, Calif.). Thermal cycle conditions were initially 50° C. for 2 min and 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 sec and 60° C. for 1 min. PCR data was analyzed according to manufacturer's specifications (User Bulletin #2 from Applied Biosystems for ABI Prism 7700 Sequence Detection System).

Conventional PCR. Conventional PCR was performed using an ABI PRISM 7700 (Perkin Elmer Applied Biosystems, Boston, Mass., USA) to confirm the results from real-time PCR. PCR was performed using 2 microliters of cDNA solution, 1×AmpliTaq Gold universal mix PCR reaction buffer (Applied Biosystems, Foster City, Calif.) and initial denaturation at 94° C. for 5 minutes. Amplification was optimized for each primer set. For IL-8, CXC ligand 3, and reticulon (94° C. for 15 seconds, 55° C. for 15 seconds and 72° C. for 30 seconds for 30 cycles); for renin (94° C. for 15 seconds, 53° C. for 15 seconds and 72° C. for 30 seconds for 38 cycles); for oxidized LDL receptor and GAPDH (94° C. for 15 seconds, 55° C. for 15 seconds and 72° C. for 30 seconds for 33 cycles). Primers used for amplification are listed in Table 1. Primer concentration in the final PCR reaction was 1 micromolar except for GAPDH, which was 0.5 micromolar. GAPDH primers were the same as real-time PCR, except that the manufacturer's TaqMan probe was not added to the final PCR reaction. Samples were run on 2% (w/v) agarose gel and stained with ethidium bromide (Sigma, St. Louis, Mo.). Images were captured using a 667 Universal Twinpack film (VWR International, South Plainfield, N.J.) using a focal-length Polaroid camera (VWR International, South Plainfield, N.J.).

TABLE 10-1 Primers used Primer name Primers Oxidized LDL S: 5′-GAGAAATCCAAAGAGCAAATGG-3′ receptor (SEQ ID NO: 1) A: 5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO: 2) Renin S: 5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO: 3) A: 5′-GAATTCTCGGAATCTCTGTTG-3′ (SEQ ID NO: 4) Reticulon S: 5′-TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO: 5) A: 5′-AGTAAACATTGAAACCACAGCC-3′ (SEQ ID NO: 6) Interleukin-8 S: 5′-TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO: 7) A: 5′-CTTCAAAAACTTCTCCACAACC-3′ (SEQ ID NO: 8) Chemokine(CXC) S: 5′-CCCACGCCACGCTCTCC-3′ ligand 3 (SEQ ID NO: 9) A: 5′-TCCTGTCAGTTGGTGCTCC-3′ (SEQ ID NO: 10)

Immunofluorescence. Derived cells were fixed with cold 4% (w/v) paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) for 10 minutes at room temperature. One isolate each of umbilicus- and placenta-derived cells at passage 0 (P0) (directly after isolation) and passage 11 (P11) (two isolates of placenta-derived, two isolates of umbilicus-derived cells) and fibroblasts (P11) were used. Immunocytochemistry was performed using antibodies directed against the following epitopes:vimentin (1:500, Sigma, St. Louis, Mo.), desmin (1:150; Sigma—raised against rabbit; or 1:300; Chemicon, Temecula, Calif.—raised against mouse,), alpha-smooth muscle actin (SMA; 1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF; 1:200; Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation, Carpinteria, Calif.). In addition, the following markers were tested on passage 11 postpartum cells: anti-human GRO alpha—PE (1:100; Becton Dickinson, Franklin Lakes, N.J.), anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.), anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa Cruz Biotech), and anti-human NOGA-A (1:100; Santa Cruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens. Where the epitope of interest was located on the cell surface (CD34, ox-LDL R1), Triton X-100 was omitted in all steps of the procedure in order to prevent epitope loss. Furthermore, in instances where the primary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v) donkey serum was used in place of goat serum throughout. Primary antibodies, diluted in blocking solution, were then applied to the cultures for a period of 1 hour at room temperature. The primary antibody solutions were removed and the cultures were washed with PBS prior to application of secondary antibody solutions (1 hour at room temperature) containing block along with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbit IgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG—FITC (1:150, Santa Cruz Biotech). Cultures were then washed and 10 micromolar DAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using an appropriate fluorescence filter on an Olympus inverted epi-fluorescent microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented fluorescence signal above control staining where the entire procedure outlined above was followed with the exception of application of a primary antibody solution. Representative images were captured using a digital color video camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, each image was taken using only one emission filter at a time. Layered montages were then prepared using Adobe Photoshop software (Adobe, San Jose, Calif.).

Preparation of Cells for FACS Analysis. Adherent Cells in Flasks were Washed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco, Carlsbad, Calif.). Cells were harvested, centrifuged, and re-suspended 3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter. One hundred microliter aliquots were delivered to conical tubes. Cells stained for intracellular antigens were permeablized with Perm/Wash buffer (BD Pharmingen, San Diego, Calif.). Antibody was added to aliquots as per manufactures specifications and the cells were incubated for in the dark for 30 minutes at 4° C. After incubation, cells were washed with PBS and centrifuged to remove excess antibody. Cells requiring a secondary antibody were resuspended in 100 microliters of 3% FBS. Secondary antibody was added as per manufactures specification and the cells were incubated in the dark for 30 minutes at 4° C. After incubation, cells were washed with PBS and centrifuged to remove excess secondary antibody. Washed cells were resuspended in 0.5 milliliters PBS and analyzed by flow cytometry. The following antibodies were used: oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BD Pharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685 and M-5284; Sigma), Donkey against Goat IgG (sc-3743; Santa Cruz, Biotech.). Flow cytometry analysis was performed with FACScalibur (Becton Dickinson San Jose, Calif.).

Results

Results of real-time PCR for selected “signature” genes performed on cDNA from cells derived from human placenta, adult and neonatal fibroblasts and Mesenchymal Stem Cells (MSCs) indicate that both oxidized LDL receptor and renin were expressed at higher level in the placenta-derived cells as compared to other cells. The data obtained from real-time PCR were analyzed by the ΔΔCT method and expressed on a logarithmic scale. Levels of reticulon and oxidized LDL receptor expression were higher in umbilicus-derived cells as compared to other cells. No significant difference in the expression levels of CXC ligand 3 and GCP-2 were found between placenta-derived cells and controls. The results of real-time PCR were confirmed by conventional PCR. Sequencing of PCR products further validated these observations. No significant difference in the expression level of CXC ligand 3 was found between placenta-derived cells and controls using conventional PCR CXC ligand 3 primers listed above.

The production of the cytokine, IL-8 in postpartum was elevated in both Growth Medium-cultured and serum-starved derived cells. All real-time PCR data was validated with conventional PCR and by sequencing PCR products.

When supernatants of cells grown in serum-free medium were examined for the presence of IL-8, the highest amounts were detected in media derived from umbilical cells and some isolates of placenta cells (Table 10-1). No IL-8 was detected in medium derived from human dermal fibroblasts.

TABLE 10-1 IL-8 protein amount measured by ELISA Cell type IL-8 hFibro ND Placenta Isolate 1 ND Umb Isolate 1 2058.42 ± 144.67 Placenta Isolate 2 ND Umb Isolate 2 2368.86 ± 22.73  Placenta Isolate3 (normal O₂) 17.27 ± 8.63 Placenta Isolate 3 (low O₂, W/O BME) 264.92 ± 9.88  Results of the ELISA assay for interleukin-8 (IL-8) performed on placenta- and umbilicus-derived cells as well as human skin fibroblasts. Values are presented here are picograms/million cells, n = 2, sem. ND: Not Detected

Placenta-derived cells were also examined for the production of oxidized LDL receptor, GCP-2 and GROalpha by FACS analysis. Cells tested positive for GCP-2. Oxidized LDL receptor and GRO were not detected by this method.

Placenta-derived cells were also tested for the production of selected proteins by immunocytochemical analysis. Immediately after isolation (passage 0), cells derived from the human placenta were fixed with 4% paraformaldehyde and exposed to antibodies for six proteins: von Willebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscle actin, and vimentin. Cells stained positive for both alpha-smooth muscle actin and vimentin. This pattern was preserved through passage 11. Only a few cells (<5%) at passage 0 stained positive for cytokeratin 18.

Cells derived from the human umbilical cord at passage 0 were probed for the production of selected proteins by immunocytochemical analysis. Immediately after isolation (passage 0), cells were fixed with 4% paraformaldehyde and exposed to antibodies for six proteins: von Willebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscle actin, and vimentin. Umbilicus-derived cells were positive for alpha-smooth muscle actin and vimentin, with the staining pattern consistent through passage 11.

Summary. Concordance between gene expression levels measured by microarray and PCR (both real-time and conventional) has been established for four genes: oxidized LDL receptor 1, renin, reticulon, and IL-8. The expression of these genes was differentially regulated at the mRNA level in the umbilical tissue- and placenta-derived cells, with IL-8 also differentially regulated at the protein level. The presence of oxidized LDL receptor was not detected at the protein level by FACS analysis in cells derived from the placenta. Differential expression of GCP-2 and CXC ligand 3 was not confirmed at the mRNA level, however GCP-2 was detected at the protein level by FACS analysis in the placenta-derived cells. Although this result is not reflected by data originally obtained from the microarray experiment, this may be due to a difference in the sensitivity of the methodologies.

Immediately after isolation (passage 0), cells derived from the human placenta stained positive for both alpha-smooth muscle actin and vimentin. This pattern was also observed in cells at passage 11. These results suggest that vimentin and alpha-smooth muscle actin expression may be preserved in cells with passaging, in the Growth Medium and under the conditions utilized in these procedures. Cells derived from the human umbilical cord at passage 0 were probed for the expression of alpha-smooth muscle actin and vimentin, and were positive for both. The staining pattern was preserved through passage 11.

Example 11 In vitro Immunological Evaluation of Umbilical Cord Tissue and Placenta-Derived Cells

UTC cells were evaluated in vitro for their immunological characteristics in an effort to predict the immunological response, if any, these cells would elicit upon in vivo transplantation. UTCs were assayed by flow cytometry for the presence of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed by antigen-presenting cells (APC) and are required for the direct stimulation of naïve CD4⁺ T cells (Abbas & Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171). The cell lines were also analyzed by flow cytometry for the expression of HLA-G (Abbas & Lichtman, 2003, supra), CD 178 (Coumans, et al., (1999) Journal of Immunological Methods 224, 185-196), and PD-L2 (Abbas & Lichtman, 2003, supra; Brown, et. al. (2003) The Journal of Immunology 170, 1257-1266). The expression of these proteins by cells residing in placental tissues is thought to mediate the immuno-privileged status of placental tissues in utero. To predict the extent to which placenta- and umbilicus-derived cell lines elicit an immune response in vivo, the cell lines were tested in a one-way mixed lymphocyte reaction (MLR).

Materials and Methods

Cell culture. Cells were cultured to confluence in Growth Medium containing penicillin/streptomycin in T75 flasks (Corning, Corning, N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.).

Antibody Staining. Cells were washed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco, Carlsbad, Mo.). Cells were harvested, centrifuged, and re-suspended in 3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter. Antibody (Table 11-1) was added to one hundred microliters of cell suspension as per manufacturer's specifications and incubated in the dark for 30 minutes at 4° C. After incubation, cells were washed with PBS and centrifuged to remove unbound antibody. Cells were re-suspended in five hundred microliters of PBS and analyzed by flow cytometry using a FACS Calibur instrument (Becton Dickinson, San Jose, Calif.).

TABLE 11-1 Antibodies Catalog Antibody Manufacturer Number HLA-DRDPDQ BD Pharmingen (San Diego, CA) 555558 CD80 BD Pharmingen (San Diego, CA) 557227 CD86 BD Pharmingen (San Diego, CA) 555665 B7-H2 BD Pharmingen (San Diego, CA) 552502 HLA-G Abcam (Cambridgeshire, UK) ab 7904-100 CD 178 Santa Cruz (San Cruz, CA) sc-19681 PD-L2 BD Pharmingen (San Diego, CA) 557846 Mouse IgG2a Sigma (St. Louis, MO) F-6522 Mouse IgG1kappa Sigma (St. Louis, MO) P-4685

Mixed Lymphocyte Reaction. Cryopreserved vials of passage 10 umbilicus-derived cells labeled as cell line A and passage 11 placenta-derived cells labeled as cell line B were sent on dry ice to CTBR (Senneville, Quebec) to conduct a mixed lymphocyte reaction using CTBR SOP No. CAC-031. Peripheral blood mononuclear cells (PBMCs) were collected from multiple male and female volunteer donors. Stimulator (donor) allogeneic PBMC, autologous PBMC, and umbilical and placental cell lines were treated with mitomycin C. Autologous and mitomycin C-treated stimulator cells were added to responder (recipient) PBMCs and cultured for 4 days. After incubation, [³H]thymidine was added to each sample and cultured for 18 hours. Following harvest of the cells, radiolabeled DNA was extracted, and [³H]-thymidine incorporation was measured using a scintillation counter.

The stimulation index for the allogeneic donor (SIAD) was calculated as the mean proliferation of the receiver plus mitomycin C-treated allogeneic donor divided by the baseline proliferation of the receiver. The stimulation index of the UTCs was calculated as the mean proliferation of the receiver plus mitomycin C-treated cell line divided by the baseline proliferation of the receiver.

Results

Mixed lymphocyte reaction—placenta-derived cells. Seven human volunteer blood donors were screened to identify a single allogeneic donor that would exhibit a robust proliferation response in a mixed lymphocyte reaction with the other six blood donors. This donor was selected as the allogeneic positive control donor. The remaining six blood donors were selected as recipients. The allogeneic positive control donor and placenta-derived cell lines were treated with mitomycin C and cultured in a mixed lymphocyte reaction with the six individual allogeneic receivers. Reactions were performed in triplicate using two cell culture plates with three receivers per plate (Table 11-2). The average stimulation index ranged from 1.3 (plate 2) to 3 (plate 1) and the allogeneic donor positive controls ranged from 46.25 (plate 2) to 279 (plate 1) (Table 11-3).

TABLE 11-2 Mixed Lymphocyte Reaction Data - Cell Line B (Placenta) DPM for Proliferation Assay Analytical Culture Replicates number System 1 2 3 Mean SD CV Plate ID: Plate 1 IM03-7769 Proliferation baseline of receiver 79 119 138 112.0 30.12 26.9 Control of autostimulation (Mitomycin C treated 241 272 175 229.3 49.54 21.6 autologous cells) MLR allogenic donor IM03-7768 (Mitomycin C treated) 23971 22352 20921 22414.7 1525.97 6.8 MLR with cell line (Mitomycin C treated cell type B) 664 559 1090 771.0 281.21 36.5 SI (donor) 200 SI (cell line) 7 IM03-7770 Proliferation baseline of receiver 206 134 262 200.7 64.17 32.0 Control of autostimulation (Mitomycin C treated 1091 602 524 739.0 307.33 41.6 autologous cells) MLR allogenic donor IM03-7768 (Mitomycin C treated) 45005 43729 44071 44268.3 660.49 1.5 MLR with cell line (Mitomycin C treated cell type B) 533 2582 2376 1830.3 1128.24 61.6 SI (donor) 221 SI (cell line) 9 IM03-7771 Proliferation baseline of receiver 157 87 128 124.0 35.17 28.4 Control of autostimulation (Mitomycin C treated 293 138 508 313.0 185.81 59.4 autologous cells) MLR allogenic donor IM03-7768 (Mitomycin C treated) 24497 34348 31388 30077.7 5054.53 16.8 MLR with cell line (Mitomycin C treated cell type B) 601 643 a 622.0 29.70 4.8 SI (donor) 243 SI (cell line) 5 IM03-7772 Proliferation baseline of receiver 56 98 51 68.3 25.81 37.8 Control of autostimulation (Mitomycin C treated 133 120 213 155.3 50.36 32.4 autologous cells) MLR allogenic donor IM03-7768 (Mitomycin C treated) 14222 20076 22168 18822.0 4118.75 21.9 MLR with cell line (Mitomycin C treated cell type B) a a a a a a SI (donor) 275 SI (cell line) a IM03-7768 Proliferation baseline of receiver 84 242 208 178.0 83.16 46.7 (allogenic donor) Control of autostimulation (Mitomycin treated 361 617 304 427.3 166.71 39.0 autologous cells) Cell line type B Proliferation baseline of receiver 126 124 143 131.0 10.44 8.0 Control of autostimulation (Mitomycin treated 822 1075 487 794.7 294.95 37.1 autologous cells) Plate ID: Plate 2 IM03-7773 Proliferation baseline of receiver 908 181 330 473.0 384.02 81.2 Control of autostimulation (Mitomycin C treated 269 405 572 415.3 151.76 36.5 autologous cells) MLR allogenic donor IM03-7768 (Mitomycin C treated) 29151 28691 28315 28719.0 418.70 1.5 MLR with cell line (Mitomycin C treated cell type B) 567 732 905 734.7 169.02 23.0 SI (donor) 61 SI (cell line) 2 IM03-7774 Proliferation baseline of receiver 893 1376 185 818.0 599.03 73.2 Control of autostimulation (Mitomycin C treated 261 381 568 403.3 154.71 38.4 autologous cells) MLR allogenic donor IM03-7768 (Mitomycin C treated) 53101 42839 48283 48074.3 5134.18 10.7 MLR with cell line (Mitomycin C treated cell type B) 515 789 294 532.7 247.97 46.6 SI (donor) 59 SI (cell line) 1 IM03-7775 Proliferation baseline of receiver 1272 300 544 705.3 505.69 71.7 Control of autostimulation (Mitomycin C treated 232 199 484 305.0 155.89 51.1 autologous cells) MLR allogenic donor IM03-7768 (Mitomycin C treated) 23554 10523 28965 21014.0 9479.74 45.1 MLR with cell line (Mitomycin C treated cell type B) 768 924 563 751.7 181.05 24.1 SI (donor) 30 SI (cell line) 1 IM03-7776 Proliferation baseline of receiver 1530 137 1046 904.3 707.22 78.2 Control of autostimulation (Mitomycin C treated 420 218 394 344.0 109.89 31.9 autologous cells) MLR allogenic donor IM03-7768 (Mitomycin C treated) 28893 32493 34746 32044.0 2952.22 9.2 MLR with cell line (Mitomycin C treated cell type B) a a a a a a SI (donor) 35 SI (cell line) a

TABLE 11-3 Average stimulation index of placenta cells and an allogeneic donor in a mixed lymphocyte reaction with six individual allogeneic receivers Average Stimulation Index Recipient Placenta Plate 1 (receivers 1-3) 279 3 Plate 2 (receivers 4-6) 46.25 1.3

Mixed lymphocyte reaction—umbilicus-derived cells. Six human volunteer blood donors were screened to identify a single allogeneic donor that will exhibit a robust proliferation response in a mixed lymphocyte reaction with the other five blood donors. This donor was selected as the allogeneic positive control donor. The remaining five blood donors were selected as recipients. The allogeneic positive control donor and placenta cell lines were mitomycin C-treated and cultured in a mixed lymphocyte reaction with the five individual allogeneic receivers. Reactions were performed in triplicate using two cell culture plates with three receivers per plate (Table 11-4). The average stimulation index ranged from 6.5 (plate 1) to 9 (plate 2) and the allogeneic donor positive controls ranged from 42.75 (plate 1) to 70 (plate 2) (Table 11-5).

TABLE 11-4 Mixed Lymphocyte Reaction Data - Cell Line A (Umbilicus) DPM for Proliferation Assay Analytical Culture Replicates number System 1 2 3 Mean SD CV Plate ID: Plate 1 IM04-2478 Proliferation baseline of receiver 1074 406 391 623.7 390.07 62.5 Control of autostimulation (Mitomycin C treated autologous cells) 672 510 1402 861.3 475.19 55.2 MLR allogenic donor IM04-2477 (Mitomycin C treated) 43777 48391 38231 43466.3 5087.12 11.7 MLR with cell line (Mitomycin C treated cell type A) 2914 5622 6109 4881.7 1721.36 35.3 SI (donor) 70 SI (cell line) 8 IM04-2479 Proliferation baseline of receiver 530 508 527 521.7 11.93 2.3 Control of autostimulation (Mitomycin C treated autologous cells) 701 567 1111 793.0 283.43 35.7 MLR allogenic donor IM04-2477 (Mitomycin C treated) 25593 24732 22707 24344.0 1481.61 6.1 MLR with cell line (Mitomycin C treated cell type A) 5086 3932 1497 3505.0 1832.21 52.3 SI (donor) 47 SI (cell line) 7 IM04-2480 Proliferation baseline of receiver 1192 854 1330 1125.3 244.90 21.8 Control of autostimulation (Mitomycin C treated autologous cells) 2963 993 2197 2051.0 993.08 48.4 MLR allogenic donor IM04-2477 (Mitomycin C treated) 25416 29721 23757 26298.0 3078.27 11.7 MLR with cell line (Mitomycin C treated cell type A) 2596 5076 3426 3699.3 1262.39 34.1 SI (donor) 23 SI (cell line) 3 IM04-2481 Proliferation baseline of receiver 695 451 555 567.0 122.44 21.6 Control of autostimulation (Mitomycin C treated autologous cells) 738 1252 464 818.0 400.04 48.9 MLR allogenic donor IM04-2477 (Mitomycin C treated) 13177 24885 15444 17835.3 6209.52 34.8 MLR with cell line (Mitomycin C treated cell type A) 4495 3671 4674 4280.0 534.95 12.5 SI (donor) 31 SI (cell line) 8 Plate ID: Plate 2 IM04-2482 Proliferation baseline of receiver 432 533 274 413.0 130.54 31.6 Control of autostimulation (Mitomycin C treated autologous cells) 1459 633 598 896.7 487.31 54.3 MLR allogenic donor IM04-2477 (Mitomycin C treated) 24286 30823 31346 28818.3 3933.82 13.7 MLR with cell line (Mitomycin C treated cell type A) 2762 1502 6723 3662.3 2724.46 74.4 SI (donor) 70 SI (cell line) 9 IM04-2477 Proliferation baseline of receiver 312 419 349 360.0 54.34 15.1 (allogenic Control of autostimulation (Mitomycin treated autologous cells) 567 604 374 515.0 123.50 24.0 donor) Cell line Proliferation baseline of receiver 5101 3735 2973 3936.3 1078.19 27.4 type A Control of autostimulation (Mitomycin treated autologous cells) 1924 4570 2153 2882.3 1466.04 50.9

TABLE 11-5 Average stimulation index of umbilicus-derived cells and an allogeneic donor in a mixed lymphocyte reaction with five individual allogeneic receivers. Average Stimulation Index Recipient Umbilicus Plate 1 (receivers 1-4) 42.75 6.5 Plate 2 (receiver 5) 70 9

Antigen presenting cell markers—placenta-derived cells. Histograms of placenta-derived cells analyzed by flow cytometry show negative expression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as noted by fluorescence value consistent with the IgG control, indicating that placental cell lines lack the cell surface molecules required to directly stimulate CD4⁺ T cells.

Immunomodulating markers—placenta-derived cells. Histograms of placenta-derived cells analyzed by flow cytometry show positive expression of PD-L2, as noted by the increased value of fluorescence relative to the IgG control, and negative expression of CD178 and HLA-G, as noted by fluorescence value consistent with the IgG control.

Antigen presenting cell markers—umbilicus-derived cells. Histograms of umbilicus-derived cells analyzed by flow cytometry show negative expression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as noted by fluorescence value consistent with the IgG control, indicating that umbilical cell lines lack the cell surface molecules required to directly stimulate CD4⁺ T cells.

Immunomodulating cell markers—umbilicus-derived cells. Histograms of umbilicus-derived cells analyzed by flow cytometry show positive expression of PD-L2, as noted by the increased value of fluorescence relative to the IgG control, and negative expression of CD178 and HLA-G, as noted by fluorescence value consistent with the IgG control.

Summary. In the mixed lymphocyte reactions conducted with placenta-derived cell lines, the average stimulation index ranged from 1.3 to 3, and that of the allogeneic positive controls ranged from 46.25 to 279. In the mixed lymphocyte reactions conducted with umbilicus-derived cell lines the average stimulation index ranged from 6.5 to 9, and that of the allogeneic positive controls ranged from 42.75 to 70. Placenta- and umbilicus-derived cell lines were negative for the expression of the stimulating proteins HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, as measured by flow cytometry. Placenta- and umbilicus-derived cell lines were negative for the expression of immuno-modulating proteins HLA-G and CD 178 and positive for the expression of PD-L2, as measured by flow cytometry. Allogeneic donor PBMCs contain antigen-presenting cells expressing HLA-DR, DQ, CD8, CD86, and B7-H2, thereby allowing for the stimulation of naïve CD4⁺ T cells. The absence of antigen-presenting cell surface molecules on placenta- and umbilicus-derived cells required for the direct stimulation of naïve CD4⁺ T cells and the presence of PD-L2, an immunomodulating protein, may account for the low stimulation index exhibited by these cells in a MLR as compared to allogeneic controls.

Example 12 Secretion of Trophic Factors by Umbilical Cord Tissue and Placental-Derived Cells

The secretion of selected trophic factors from placenta- and umbilicus-derived cells was measured. Factors selected for detection included: (1) those known to have angiogenic activity, such as hepatocyte growth factor (HGF) (Rosen et al. (1997) Ciba Found. Symp. 212:215-26), monocyte chemotactic protein 1 (MCP-1) (Salcedo et al. (2000) Blood 96; 34-40), interleukin-8 (IL-8) (Li et al. (2003) J. Immunol. 170:3369-76), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77:812-8), matrix metalloproteinase 1 (TIMP1), angiopoietin 2 (ANG2), platelet derived growth factor (PDGF-bb), thrombopoietin (TPO), heparin-binding epidermal growth factor (HB-EGF), stromal-derived factor 1alpha (SDF-1 alpha); (2) those known to have neurotrophic/neuroprotective activity, such as brain-derived neurotrophic factor (BDNF) (Cheng et al. (2003) Dev. Biol. 258; 319-33), interleukin-6 (IL-6), granulocyte chemotactic protein-2 (GCP-2), transforming growth factor beta2 (TGFbeta2); and (3) those known to have chemokine activity, such as macrophage inflammatory protein 1alpha (MIP1a), macrophage inflammatory protein 1beta (MIP1b), monocyte chemoattractant-1 (MCP-1), Rantes (regulated on activation, normal T cell expressed and secreted), 1309, thymus and activation-regulated chemokine (TARC), Eotaxin, macrophage-derived chemokine (MDC), IL-8).

Methods & Materials

Cell culture. Cells from placenta and umbilicus as well as human fibroblasts derived from human neonatal foreskin were cultured in Growth Medium with penicillin/streptomycin on gelatin-coated T75 flasks. Cells were cryopreserved at passage 11 and stored in liquid nitrogen. After thawing of the cells, Growth Medium was added to the cells followed by transfer to a 15 milliliter centrifuge tube and centrifugation of the cells at 150×g for 5 minutes. The supernatant was discarded. The cell pellet was resuspended in 4 milliliters Growth Medium, and cells were counted. Cells were seeded at 375,000 cells/75 cm² flask containing 15 milliliters of Growth Medium and cultured for 24 hours. The medium was changed to a serum-free medium (DMEM—low glucose (Gibco), 0.1% (w/v) bovine serum albumin (Sigma), penicillin/streptomycin (Gibco)) for 8 hours. Conditioned serum-free medium was collected at the end of incubation by centrifugation at 14,000×g for 5 minutes and stored at −20° C. To estimate the number of cells in each flask, cells were washed with PBS and detached using 2 milliliters trypsin/EDTA. Trypsin activity was inhibited by addition of 8 milliliters Growth Medium. Cells were centrifuged at 150×g for 5 minutes. Supernatant was removed, and cells were resuspended in 1 milliliter Growth Medium. Cell number was estimated using a hemocytometer.

ELISA assay. Cells were grown at 37° C. in 5% carbon dioxide and atmospheric oxygen. Placenta-derived cells (batch 101503) also were grown in 5% oxygen or beta-mercaptoethanol (BME). The amount of MCP-1, IL-6, VEGF, SDF-1alpha, GCP-2, IL-8, and TGF-beta 2 produced by each cell sample was measured by an ELISA assay (R&D Systems, Minneapolis, Minn.). All assays were performed according to the manufacturer's instructions.

Searchlight™ multiplexed ELISA assay. Chemokines (MIP1a, MIP1b, MCP-1, Rantes, 1309, TARC, Eotaxin, MDC, IL8), BDNF, and angiogenic factors (HGF, KGF, bFGF, VEGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF were measured using SearchLight Proteome Arrays (Pierce Biotechnology Inc.). The Proteome Arrays are multiplexed sandwich ELISAs for the quantitative measurement of two to 16 proteins per well. The arrays are produced by spotting a 2×2, 3×3, or 4×4 pattern of four to 16 different capture antibodies into each well of a 96-well plate. Following a sandwich ELISA procedure, the entire plate is imaged to capture chemiluminescent signal generated at each spot within each well of the plate. The amount of signal generated in each spot is proportional to the amount of target protein in the original standard or sample.

Results

ELISA assay. MCP-1 and IL-6 were secreted by placenta- and umbilicus-derived cells and dermal fibroblasts (Table 12-1). SDF-1alpha was secreted by placenta-derived cells cultured in 5% O₂ and by fibroblasts. GCP-2 and IL-8 were secreted by umbilicus-derived cells and by placenta-derived cells cultured in the presence of BME or 5% O₂. GCP-2 also was secreted by human fibroblasts. TGF-beta2 was not detectable by ELISA assay.

TABLE 12-1 ELISA assay results (values presented are picograms/milliliter/million cells (n = 2, sem) MCP-1 IL-6 VEGF SDF-1α GCP-2 IL-8 TGF-β2 Fibroblast 17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ± 1 ND ND Placenta (042303) 60 ± 3 41 ± 2 ND ND ND ND ND Umbilicus (022803) 1150 ± 74  4234 ± 289 ND ND 160 ± 11 2058 ± 145 ND Placenta (071003) 125 ± 16 10 ± 1 ND ND ND ND ND Umbilicus (071003) 2794 ± 84  1356 ± 43  ND ND 2184 ± 98  2369 ± 23  ND Placenta (101503) BME  21 ± 10 67 ± 3 ND ND 44 ± 9 17 ± 9 ND Placenta (101503) 5% O₂,  77 ± 16 339 ± 21 ND 1149 ± 137 54 ± 2 265 ± 10 ND W/O BME Key: ND: Not Detected.

Searchlight™ multiplexed ELISA assay. TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, 1309, TARC, MDC, and IL-8 were secreted from umbilicus-derived cells (Tables 12-2 and 12-3). TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were secreted from placenta-derived cells (Tables 12-2 and 12-3). No Ang2, VEGF, or PDGF-bb were detected.

TABLE 12-2 Searchlight ™ Multiplexed ELISA assay results TIMP1 ANG2 PDGFbb TPO KGF HGF FGF VEGF HBEGF BDNF Hfb 19306.3 ND ND 230.5 5.0 ND ND 27.9 1.3 ND P1 24299.5 ND ND 546.6 8.8 16.4 ND ND 3.81.3 ND U1 57718.4 ND ND 1240.0 5.8 559.3 148.7 ND 9.3 165.7 P3 14176.8 ND ND 568.7 5.2 10.2 ND ND 1.9 33.6 U3 21850.0 ND ND 1134.5 9.0 195.6  30.8 ND 5.4 388.6 Key: hFB (human fibroblasts), P1 (placenta-derived cells (042303)), U1 (umbilicus-derived cells (022803)), P3 (placenta-derived cells (071003)), U3 (umbilicus-derived cells (071003)). ND: Not Detected.

TABLE 12-3 Searchlight ™ Multiplexed ELISA assay results MIP1a MIP1b MCP1 RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND 204.9 P1 79.5 ND 228.4  4.1 ND 3.8 12.2 ND 413.5 U1 ND 8.0 1694.2 ND 22.4 37.6 ND 18.9 51930.1 P3 ND ND 102.7 ND ND 0.4 ND ND 63.8 U3 ND 5.2 2018.7 41.5 11.6 21.4 ND  4.8 10515.9 Key: hFB (human fibroblasts), P1 (placenta-derived cells (042303)), U1 (umbilicus-derived cells (022803)), P3 (placenta-derived cells (071003)), U3 (umbilicus-derived cells (071003)). ND: Not Detected.

Summary. Umbilicus- and placenta-derived cells secreted a number of trophic factors. Some of these trophic factors, such as HGF, bFGF, MCP-1 and IL-8, play important roles in angiogenesis. Other trophic factors, such as BDNF and IL-6, have important roles in neural regeneration.

Example 13 Short-Term Neural Differentiation of Umbilical Cord Tissue and Placenta-Derived Cells

The ability of placenta- and umbilicus-derived cells to differentiate into neural lineage cells was examined.

Materials & Methods

Isolation and Expansion of Umbilicus- and Placenta-derived Cells. Cells from placental and umbilical tissues were isolated and expanded as described in Example 1.

Modified Woodbury-Black Protocol. (A) This assay was adapted from an assay originally performed to test the neural induction potential of bone marrow stromal cells (1). Umbilicus-derived cells (022803) P4 and placenta-derived cells (042203) P3 were thawed and culture expanded in Growth Media at 5,000 cells/cm² until sub-confluence (75%) was reached. Cells were then trypsinized and seeded at 6,000 cells per well of a Titretek II glass slide (VWR International, Bristol, Conn.). As controls, mesenchymal stem cells (P3; 1F2155; Cambrex, Walkersville, Md.), osteoblasts (P5; CC2538; Cambrex), adipose-derived cells (Artecel, U.S. Pat. No. 6,555,374 B1) (P6; Donor 2) and neonatal human dermal fibroblasts (P6; CC2509; Cambrex) were also seeded under the same conditions.

All cells were initially expanded for 4 days in DMEM/F12 medium (Invitrogen, Carlsbad, Calif.) containing 15% (v/v) fetal bovine serum (FBS; Hyclone, Logan, Utah), basic fibroblast growth factor (bFGF; 20 nanograms/milliliter; Peprotech, Rocky Hill, N.J.), epidermal growth factor (EGF; 20 nanograms/milliliter; Peprotech) and penicillin/streptomycin (Invitrogen). After four days, cells were rinsed in phosphate-buffered saline (PBS; Invitrogen) and were subsequently cultured in DMEM/F12 medium+20% (v/v) FBS+penicillin/streptomycin for 24 hours. After 24 hours, cells were rinsed with PBS. Cells were then cultured for 1-6 hours in an induction medium which was comprised of DMEM/F12 (serum-free) containing 200 mM butylated hydroxyanisole, 10 μM potassium chloride, 5 milligram/milliliter insulin, 10 μM forskolin, 4 μM valproic acid, and 2 μM hydrocortisone (all chemicals from Sigma, St. Louis, Mo.). Cells were then fixed in 100% ice-cold methanol and immunocytochemistry was performed (see methods below) to assess human nestin protein expression.

(B) Derived cells (umbilicus (022803) P11; placenta (042203) P11) and adult human dermal fibroblasts (1F1853, P11) were thawed and culture expanded in Growth Medium at 5,000 cells/cm2 until sub-confluence (75%) was reached. Cells were then trypsinized and seeded at similar density as in (A), but onto (1) 24 well tissue culture-treated plates (TCP, Falcon brand, VWR International), (2) TCP wells+2% (w/v) gelatin adsorbed for 1 hour at room temperature, or (3) TCP wells+20 μg/milliliter adsorbed mouse laminin (adsorbed for a minimum of 2 hours at 37° C.; Invitrogen).

Exactly as in (A), cells were initially expanded and media switched at the aforementioned timeframes. One set of cultures was fixed, as before, at 5 days and six hours, this time with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at room temperature. In the second set of cultures, medium was removed and switched to Neural Progenitor Expansion medium (NPE) consisting of Neurobasal-A medium (Invitrogen) containing B27 (B27 supplement; Invitrogen), L-glutamine (4 mM), and penicillin/streptomycin (Invitrogen). NPE medium was further supplemented with retinoic acid (RA; 1 μM; Sigma). This medium was removed 4 days later and cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and stained for nestin, GFAP, and TuJ1 protein expression (see Table 13-1).

TABLE 13-1 Summary of Primary Antibodies Used Antibody Concentration Vendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA Human Nestin 1:100 Chemicon TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000 DakoCytomation, Carpinteria, CA Tyrosine hydroxylase (TH) 1:1000 Chemicon GABA 1:400 Chemicon Desmin (mouse) 1:300 Chemicon alpha - alpha-smooth muscle actin 1:400 Sigma Human nuclear protein (hNuc) 1:150 Chemicon

Two Stage Differentiation Protocol. Derived cells (umbilicus (042203) P11, placenta (022803) P11), adult human dermal fibroblasts (P11; 1F1853; Cambrex) were thawed and culture expanded in Growth Medium at 5,000 cells/cm² until sub-confluence (75%) was reached. Cells were then trypsinized and seeded at 2,000 cells/cm², but onto 24 well plates coated with laminin (BD Biosciences, Franklin Lakes, N.J.) in the presence of NPE media supplemented with bFGF (20 nanograms/milliliter; Peprotech, Rocky Hill, N.J.) and EGF (20 nanograms/milliliter; Peprotech) [whole media composition further referred to as NPE+F+E]. At the same time, adult rat neural progenitors isolated from hippocampus (P4; (062603) were also plated onto 24 well laminin-coated plates in NPE+F+E media. All cultures were maintained in such conditions for a period of 6 days (cells were fed once during that time) at which time media was switched to the differentiation conditions listed in Table N1-2 for an additional period of 7 days. Cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and stained for human or rat nestin, GFAP, and TuJ1 protein expression.

TABLE 13-2 Summary of Conditions for Two-Stage Differentiation Protocol A B COND. # PRE-DIFFERENTIATION 2^(nd) STAGE DIFF 1 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) 2 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) + RA (1 μM) 3 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + RA (1 μM) 4 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + F (20 ng/ml) + E (20 ng/ml) 5 NPE + F (20 ng/ml) + E (20 ng/ml) Growth Medium 6 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 1B + MP52 (20 ng/ml) 7 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 1B + BMP7 (20 ng/ml) 8 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 1B + GDNF (20 ng/ml) 9 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 2B + MP52 (20 ng/ml) 10 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 2B + BMP7 (20 ng/ml) 11 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 2B + GDNF (20 ng/ml) 12 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 3B + MP52 (20 ng/ml) 13 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 3B + BMP7 (20 ng/ml) 14 NPE + F (20 ng/ml) + E (20 ng/ml) Condition 3B + GDNF (20 ng/ml) 15 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + MP52 (20 ng/ml) 16 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + BMP7 (20 ng/ml) 17 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + GDNF (20 ng/ml)

Multiple growth factor protocol. Umbilicus-derived cells (P11; (042203)) were thawed and culture expanded in Growth Medium at 5,000 cells/cm² until sub-confluence (75%) was reached. Cells were then trypsinized and seeded at 2,000 cells/cm², onto 24 well laminin-coated plates (BD Biosciences) in the presence of NPE+F (20 nanograms/milliliter)+E (20 nanograms/milliliter). In addition, some wells contained NPE+F+E+2% FBS or 10% FBS. After four days of “pre-differentiation” conditions, all media were removed and samples were switched to NPE medium supplemented with sonic hedgehog (SHH; 200 nanograms/milliliter; Sigma, St. Louis, Mo.), FGF8 (100 nanograms/milliliter; Peprotech), BDNF (40 nanograms/milliliter; Sigma), GDNF (20 nanograms/milliliter; Sigma), and retinoic acid (1 μM; Sigma). Seven days post medium change, cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and stained for human nestin, GFAP, TuJ1, desmin, and alpha-smooth muscle actin expression.

Neural progenitor co-culture protocol. Adult rat hippocampal progenitors (062603) were plated as neurospheres or single cells (10,000 cells/well) onto laminin-coated 24 well dishes (BD Biosciences) in NPE+F (20 nanograms/milliliter)+E (20 nanograms/milliliter).

Separately, umbilicus-derived cells (042203) P11 and placenta-derived cells (022803) P11 were thawed and culture expanded in NPE+F (20 nanograms/milliliter)+E (20 nanograms/milliliter) at 5,000 cells/cm² for a period of 48 hours. Cells were then trypsinized and seeded at 2,500 cells/well onto existing cultures of neural progenitors. At that time, existing medium was exchanged for fresh medium. Four days later, cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at room temperature, and stained for human nuclear protein (hNuc; Chemicon) (Table NU1-1 above) to identify the umbilical cord tissue- and placenta-derived cells.

Immunocytochemistry. Immunocytochemistry was performed using the antibodies listed in Table NU1-1. Cultures were washed with phosphate-buffered saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes to access intracellular antigens. Primary antibodies, diluted in blocking solution, were then applied to the cultures for a period of 1 hour at room temperature. Next, primary antibodies solutions were removed and cultures washed with PBS prior to application of secondary antibody solutions (1 hour at room temperature) containing blocking solution along with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; Molecular Probes). Cultures were then washed and 10 micromolar DAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using the appropriate fluorescence filter on an Olympus inverted epi-fluorescent microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented fluorescence signal above control staining where the entire procedure outlined above was followed with the exception of application of a primary antibody solution. Representative images were captured using a digital color video camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, each image was taken using only one emission filter at a time. Layered montages were then prepared using Adobe Photoshop software (Adobe, San Jose, Calif.).

Results

Woodbury-Black protocol. (Woodbury, D. et al. (2000). J. Neurosci. Research. 61(4): 364-70). (A) Upon incubation in this neural induction composition, all cell types transformed into cells with bipolar morphologies and extended processes. Other larger non-bipolar morphologies were also observed. Furthermore, the induced cell populations stained positively for nestin, a marker of multipotent neural stem and progenitor cells.

(B) When repeated on tissue culture plastic (TCP) dishes, nestin expression was not observed unless laminin was pre-adsorbed to the culture surface. To further assess whether nestin-expressing cells could then go on to generate mature neurons, the umbilical cord tissue derived-cells, placenta-derived cells and fibroblasts were exposed to NPE+RA (1 μM), a media composition known to induce the differentiation of neural stem and progenitor cells into such cells (Jang, Y. K. et al. (2004). J. Neurosci. Research. 75(4): 573-84; Jones-Villeneuve, E. M. et al. (1983). Mol Cel Biol. 3(12): 2271-9; Mayer-Proschel, M. et al. (1997). Neuron. 19(4): 773-85). Cells were stained for TuJ1, a marker for immature and mature neurons, GFAP, a marker of astrocytes, and nestin. Under no conditions was TuJ1 detected, nor were cells with neuronal morphology observed, suggesting that neurons were not generated in the short term. Furthermore, nestin and GFAP were no longer expressed by umbilical and placental-derived cells, as determined by immunocytochemistry.

Two-stage differentiation. Umbilicus and placenta isolates (as well as human fibroblasts and rodent neural progenitors as negative and positive control cell types, respectively) were plated on laminin (neural promoting)-coated dishes and exposed to 13 different growth conditions (and two control conditions) known to promote differentiation of neural progenitors into neurons and astrocytes. In addition, two conditions were added to examine the influence of GDF5, and BMP7 on cell differentiation. Generally, a two-step differentiation approach was taken, where the cells were first placed in neural progenitor expansion conditions for a period of 6 days, followed by full differentiation conditions for 7 days. Morphologically, both umbilicus- and placenta-derived cells exhibited fundamental changes in cell morphology throughout the time-course of this procedure. However, neuronal or astrocytic-shaped cells were not observed except for in control, neural progenitor-plated conditions. Immunocytochemistry, negative for human nestin, TuJ1, and GFAP confirmed the morphological observations.

Multiple growth factors. Following one week's exposure to a variety of neural differentiation agents, cells were stained for markers indicative of neural progenitors (human nestin), neurons (TuJ1), and astrocytes (GFAP). Cells grown in the first stage in non-serum containing media had different morphologies than those cells in serum containing (2% or 10%) media, indicating potential neural differentiation. Specifically, following a two step procedure of exposing umbilicus-derived cells to EGF and bFGF, followed by SHH, FGF8, GDNF, BDNF, and retinoic acid, cells showed long extended processes similar to the morphology of cultured astrocytes. When 2% FBS or 10% FBS was included in the first stage of differentiation, cell number was increased and cell morphology was unchanged from control cultures at high density. Potential neural differentiation was not evidenced by immunocytochemical analysis for human nestin, TuJ1, or GFAP.

Neural progenitor and UTC co-culture. Umbilical and placental-derived cells were plated onto cultures of rat neural progenitors seeded two days earlier in neural expansion conditions (NPE+F+E). While visual confirmation of plated UTCs and placenta derived cells proved that these cells were plated as single cells, human-specific nuclear staining (hNuc) 4 days post-plating (6 days total) showed that they tended to ball up and avoid contact with the neural progenitors. Furthermore, where UTCs attached, these cells spread out and appeared to be innervated by differentiated neurons that were of rat origin, suggesting that the UTCs may have differentiated into muscle cells. This observation was based upon morphology under phase contrast microscopy. Another observation was that typically large cell bodies (larger than neural progenitors) possessed morphologies resembling neural progenitors, with thin processes spanning out in multiple directions. HNuc staining (found in one half of the cell's nucleus) suggested that in some cases these human cells may have fused with rat progenitors and assumed their phenotype. Control wells containing only neural progenitors had fewer total progenitors and apparent differentiated cells than did co-culture wells containing umbilicus or placenta derived cells, further indicating that both umbilicus- and placenta-derived cells influenced the differentiation and behavior of neural progenitors, either by release of chemokines and cytokines, or by contact-mediated effects.

Summary. Multiple protocols were conducted to determine the short term potential of UTCs and placenta derived cells to differentiate into neural lineage cells. These included phase contrast imaging of morphology in combination with immunocytochemistry for nestin, TuJ1, and GFAP, proteins associated with multipotent neural stem and progenitor cells, immature and mature neurons, and astrocytes, respectively. Evidence was observed to suggest that neural differentiation occurred in certain instances in these short-term protocols.

Several notable observations were made in co-cultures of UTCs and placenta derived cells with neural progenitors. This approach, using human UTCs and placenta derived cells along with a xenogeneic cell type allowed for absolute determination of the origin of each cell in these cultures. First, some cells were observed in these cultures where the cell cytoplasm was enlarged, with neurite-like processes extending away from the cell body, yet only half of the body labeled with hNuc protein. Those cells may have been human UTCs and placenta derived cells that had differentiated into neural lineage cells or they may have been UTCs and placenta derived cells that had fused with neural progenitors. Second, it appeared that neural progenitors extended neurites to UTCs and placenta derived cells in a way that indicates the progenitors differentiated into neurons and innervated the UTCs and placenta derived cells. Third, cultures of neural progenitors and UTCs and placenta derived cells had more cells of rat origin and larger amounts of differentiation than control cultures of neural progenitors alone, further indicating that plated UTCs and placenta derived cells provided soluble factors and or contact-dependent mechanisms that stimulated neural progenitor survival, proliferation, and/or differentiation.

Example 14 Lone-Term Neural Differentiation of Umbilical Cord Tissue and Placenta-Derived Cells

The ability of umbilicus and placenta-derived cells to undergo long-term differentiation into neural lineage cells was evaluated.

Materials & Methods

Isolation and Expansion of Cells. Cells were isolated and expanded as described in previous Examples.

Cell Thaw and Plating. Frozen aliquots of cells (umbilicus (022803) P11; (042203) P11; (071003) P12; placenta (101503) P7) previously grown in Growth Medium were thawed and plated at 5,000 cells/cm² in T-75 flasks coated with laminin (BD, Franklin Lakes, N.J.) in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement, Invitrogen), L-glutamine (4 mM), and Penicillin/Streptomycin (10 milliliters), the combination of which is herein referred to as Neural Progenitor Expansion (NPE) media. NPE media was further supplemented with bFGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referred to as NPE+bFGF+EGF.

Control Cell Plating. In addition, adult human dermal fibroblasts (P11, Cambrex, Walkersville, Md.) and mesenchymal stem cells (P5, Cambrex) were thawed and plated at the same cell seeding density on laminin-coated T-75 flasks in NPE+bFGF+EGF. As a further control, fibroblasts, umbilicus, and placenta cells were grown in Growth Medium for the period specified for all cultures.

Cell Expansion. Media from all cultures were replaced with fresh media once a week and cells observed for expansion. In general, each culture was passaged one time over a period of one month because of limited growth in NPE+bFGF+EGF.

Immunocytochemistry. After a period of one month, all flasks were fixed with cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at room temperature. Immunocytochemistry was performed using antibodies directed against TuJ1 (BIII Tubulin; 1:500; Sigma, St. Louis, Mo.) and GFAP (glial fibrillary acidic protein; 1:2000; DakoCytomation, Carpinteria, Calif.). Briefly, cultures were washed with phosphate-buffered saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes to access intracellular antigens. Primary antibodies, diluted in blocking solution, were then applied to the cultures for a period of 1 hour at room temperature. Next, primary antibodies solutions were removed and cultures washed with PBS prior to application of secondary antibody solutions (1 hour at room temperature) containing block along with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; Molecular Probes). Cultures were then washed and 10 micromolar DAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using the appropriate fluorescence filter on an Olympus inverted epi-fluorescent microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented fluorescence signal above control staining where the entire procedure outlined above was followed with the exception of application of a primary antibody solution. Representative images were captured using a digital color video camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, each image was taken using only one emission filter at a time. Layered montages were then prepared using Adobe Photoshop software (Adobe, San Jose, Calif.).

TABLE 14-1 Summary of Primary Antibodies Used Antibody Concentration Vendor TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000 DakoCytomation, Carpinteria, CA

Results

NPE+bFGF+EGF media slows proliferation of UTCs and alters their morphology. Immediately following plating, a subset of cells attached to the culture flasks coated with laminin. This may have been due to cell death as a function of the freeze/thaw process or because of the new growth conditions. Cells that did attach adopted morphologies different from those observed in Growth Media.

Upon confluence, cultures were passaged and observed for growth. Very little expansion took place of those cells that survived passage. At this point, very small cells with no spread morphology and with phase-bright characteristics began to appear in cultures of umbilicus-derived cells. These areas of the flask were followed over time. From these small cells, bifurcating processes emerged with varicosities along their lengths, features very similar to previously described PSA-NCAM+ neuronal progenitors and TuJ1+ immature neurons derived from brain and spinal cord (Mayer-Proschel, M. et al. (1997). Neuron. 19(4): 773-85; Yang, H. et al. (2000). PNAS. 97(24): 13366-71). With time, these cells became more numerous, yet still were only found in clones.

Clones of umbilicus-derived cells express neuronal proteins. Cultures were fixed at one month post-thawing/plating and stained for the neuronal protein TuJ1 and GFAP, an intermediate filament found in astrocytes. While all control cultures grown in Growth Medium and human fibroblasts and MSCs grown in NPE+bFGF+EGF medium were found to be TuJ1-/GFAP-, TuJ1 was detected in the umbilicus and placenta derived cells. Expression was observed in cells with and without neuronal-like morphologies. No expression of GFAP was observed in either culture. The percentage of cells expressing TuJ1 with neuronal-like morphologies was less than or equal to 1% of the total population (n=3 umbilicus-derived cell isolates tested). While not quantified, the percentage of TuJ1+ cells without neuronal morphologies was higher in umbilicus-derived cell cultures than placenta-derived cell cultures. These results appeared specific as age-matched controls in Growth Medium did not express TuJ1.

Summary. Methods for generating differentiated neurons (based on TuJ1 expression and neuronal morphology) from umbilicus-derived cells were developed. While expression for TuJ1 was not examined earlier than one month in vitro, it is clear that at least a small population of umbilicus-derived cells can give rise to neurons either through default differentiation or through long-term induction following one month's exposure to a minimal media supplemented with L-glutamine, basic FGF, and EGF.

Example 15 Trophic Factors for Neural Progenitor Support

The influence of umbilicus- and placenta-derived cells on adult neural stem and progenitor cell survival and differentiation through non-contact dependent (trophic) mechanisms was examined.

Materials & Methods

Adult neural stem and progenitor cell isolation. Fisher 344 adult rats were sacrificed by CO₂ asphyxiation followed by cervical dislocation. Whole brains were removed intact using bone rongeurs and hippocampus tissue dissected based on coronal incisions posterior to the motor and somatosensory regions of the brain (Paxinos, G. & Watson, C. 1997. The Rat Brain in Stereotaxic Coordinates). Tissue was washed in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement; Invitrogen), L-glutamine (4 mM; Invitrogen), and penicillin/streptomycin (Invitrogen), the combination of which is herein referred to as Neural Progenitor Expansion (NPE) medium. NPE medium was further supplemented with bFGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referred to as NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissue minced with a scalpel. Minced tissue was collected and trypsin/EDTA (Invitrogen) added as 75% of the total volume. DNAse (100 microliters per 8 milliliters total volume, Sigma, St. Louis, Mo.) was also added. Next, the tissue/media was sequentially passed through an 18 gauge needle, 20 gauge needle, and finally a 25 gauge needle one time each (all needles from Becton Dickinson, Franklin Lakes, N.J.). The mixture was centrifuged for 3 minutes at 250 g. Supernatant was removed, fresh NPE+bFGF+EGF was added and the pellet resuspended. The resultant cell suspension was passed through a 40 micrometer cell strainer (Becton Dickinson), plated on laminin-coated T-75 flasks (Becton Dickinson) or low cluster 24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGF media until sufficient cell numbers were obtained for the studies outlined.

Cell plating. Derived cells (umbilicus (022803) P12, (042103) P12, (071003) P12; placenta (042203) P12) previously grown in Growth Medium were plated at 5,000 cells/transwell insert (sized for 24 well plate) and grown for a period of one week in Growth Medium in inserts to achieve confluence.

Adult neural progenitor plating. Neural progenitors, grown as neurospheres or as single cells, were seeded onto laminin-coated 24 well plates at an approximate density of 2,000 cells/well in NPE+bFGF+EGF for a period of one day to promote cellular attachment. One day later, transwell inserts containing derived cells were added according to the following scheme:

-   -   (1) Transwell (umbilicus-derived cells in Growth Media, 200         microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)     -   (2) Transwell (placenta-derived cells in Growth Media, 200         microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)     -   (3) Transwell (adult human dermal fibroblasts) 1F1853; Cambrex,         Walkersville, Md.) P12 in Growth Media, 200 microliters)+neural         progenitors (NPE+bFGF+EGF, 1 milliliter)     -   (4) Control: neural progenitors alone (NPE+bFGF+EGF, 1         milliliter)     -   (5) Control: neural progenitors alone (NPE only, 1 milliliter)

Immunocytochemistry. After 7 days in co-culture, all conditions were fixed with cold 4% (w/v) paraformaldehyde (Sigma) for a period of 10 minutes at room temperature. Immunocytochemistry was performed using antibodies directed against the epitopes listed in Table 15-1. Briefly, cultures were washed with phosphate-buffered saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes to access intracellular antigens. Primary antibodies, diluted in blocking solution, were then applied to the cultures for a period of 1 hour at room temperature. Next, primary antibodies solutions were removed and cultures washed with PBS prior to application of secondary antibody solutions (1 hour at room temperature) containing blocking solution along with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; Molecular Probes). Cultures were then washed and 10 micromolar DAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using the appropriate fluorescence filter on an Olympus inverted epi-fluorescent microscope (Olympus, Melville, N.Y.). In all cases, positive staining represented fluorescence signal above control staining where the entire procedure outlined above was followed with the exception of application of a primary antibody solution. Representative images were captured using a digital color video camera and ImagePro software (Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, each image was taken using only one emission filter at a time. Layered montages were then prepared using Adobe Photoshop software (Adobe, San Jose, Calif.).

TABLE 15-1 Summary of Primary Antibodies Used Antibody Concentration Vendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO Tyrosine hydroxylase (TH) 1:1000 Chemicon GABA 1:400 Chemicon GFAP 1:2000 DakoCytomation, Carpinteria, CA Myelin Basic Protein (MBP) 1:400 Chemicon

Quantitative analysis of neural progenitor differentiation. Quantification of hippocampal neural progenitor differentiation was examined. A minimum of 1000 cells were counted per condition or if less, the total number of cells observed in that condition. The percentage of cells positive for a given stain was assessed by dividing the number of positive cells by the total number of cells as determined by DAPI (nuclear) staining

Mass spectrometry analysis & 2D gel electrophoresis. In order to identify unique, secreted factors as a result of co-culture, conditioned media samples taken prior to culture fixation were frozen down at −80° C. overnight. Samples were then applied to ultrafiltration spin devices (MW cutoff 30 kD). Retentate was applied to immunoaffinity chromatography (anti-Hu-albumin; IgY) (immunoaffinity did not remove albumin from the samples). Filtrate was analyzed by MALDI. The pass through was applied to Cibachron Blue affinity chromatography. Samples were analyzed by SDS-PAGE and 2D gel electrophoresis.

Results

Derived cell co-culture stimulates adult neural progenitor differentiation. Following culture with umbilicus- or placenta-derived cells, co-cultured neural progenitor cells derived from adult rat hippocampus exhibited significant differentiation along all three major lineages in the central nervous system. This effect was clearly observed after five days in co-culture, with numerous cells elaborating complex processes and losing their phase bright features characteristic of dividing progenitor cells. Conversely, neural progenitors grown alone in the absence of bFGF and EGF appeared unhealthy and survival was limited.

After completion of the procedure, cultures were stained for markers indicative of undifferentiated stem and progenitor cells (nestin), immature and mature neurons (TuJ1), astrocytes (GFAP), and mature oligodendrocytes (MBP). Differentiation along all three lineages was confirmed while control conditions did not exhibit significant differentiation as evidenced by retention of nestin-positive staining amongst the majority of cells. While both umbilicus- and placenta-derived cells induced cell differentiation, the degree of differentiation for all three lineages was less in co-cultures with placenta-derived cells than in co-cultures with umbilicus-derived cells.

The percentage of differentiated neural progenitors following co-culture with umbilicus-derived cells was quantified (Table 15-2). Umbilicus-derived cells significantly enhanced the number of mature oligodendrocytes (MBP) (24.0% vs. 0% in both control conditions). Furthermore, co-culture enhanced the number of GFAP+ astrocytes and TuJ1+ neurons in culture (47.2% and 8.7% respectively). These results were confirmed by nestin staining indicating that progenitor status was lost following co-culture (13.4% vs. 71.4% in control condition 4).

Though differentiation also appeared to be influenced by adult human fibroblasts, such cells were not able to promote the differentiation of mature oligodendrocytes nor were they able to generate an appreciable quantity of neurons. Though not quantified, fibroblasts did, however, appear to enhance the survival of neural progenitors.

TABLE 15-2 Quantification of progenitor differentiation in control vs transwell co- culture with umbilical-derived cells (E = EGF, F = bFGF) F + E/Umb F + E/F + E F + E/removed Antibody [Cond. 1] [Cond. 4] [Cond. 5] TuJ1 8.7%  2.3%  3.6% GFAP 47.2% 30.2% 10.9% MBP 23.0%   0%   0% Nestin 13.4% 71.4% 39.4%

Identification of unique compounds. Conditioned media from umbilicus- and placenta-derived co-cultures, along with the appropriate controls (NPE media±1.7% serum, media from co-culture with fibroblasts), were examined for differences. Potentially unique compounds were identified and excised from their respective 2D gels.

Summary. Co-culture of adult neural progenitor cells with umbilicus or placenta derived cells results in differentiation of those cells. Results presented in this example indicate that the differentiation of adult neural progenitor cells following co-culture with umbilicus-derived cells is particularly profound. Specifically, a significant percentage of mature oligodendrocytes was generated in co-cultures of umbilicus-derived cells. In view of the lack of contact between the umbilicus-derived cells and the neural progenitors, this result appears to be a function of soluble factors released from the umbilicus-derived cells (trophic effect).

Several other observations were made. First, there were very few cells in the control condition where EGF and bFGF were removed. Most cells died and on average, there were about 100 cells or fewer per well. Second, it is to be expected that there would be very little differentiation in the control condition where EGF and bFGF was retained in the medium throughout, since this is normally an expansion medium. While approximately 70% of the cells were observed to retain their progenitor status (nestin+), about 30% were GFAP+ (indicative of astrocytes). This may be due to the fact that such significant expansion occurred throughout the course of the procedure that contact between progenitors induced this differentiation (Song, H. et al. 2002. Nature 417(6884): 39-44).

Example 16 Transplantation of Umbilicus- and Placenta-Derived Cells

Cells derived from the umbilicus and placenta are useful for regenerative therapies. The tissue produced by umbilical cord tissue and placenta-derived cells transplanted into SCID mice with a biodegradable material was evaluated. The materials evaluated were Vicryl non-woven, 35/65 PCL/PGA foam, and RAD 16 self-assembling peptide hydrogel.

Methods & Materials

Cell Culture. Placenta- and umbilicus-derived cells were grown in Growth Medium (DMEM-low glucose (Gibco, Carlsbad Calif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.), penicillin/streptomycin (Gibco)) in a gelatin-coated flasks.

Sample Preparation. One million viable cells were seeded in 15 microliters Growth Medium onto 5 mm diameter, 2.25 mm thick Vicryl non-woven scaffolds (64.33 milligrams/cc; Lot#3547-47-1) or 5 mm diameter 35/65 PCL/PGA foam (Lot# 3415-53). Cells were allowed to attach for two hours before adding more Growth Medium to cover the scaffolds. Cells were grown on scaffolds overnight. Scaffolds without cells were also incubated in medium.

RAD16 self-assembling peptides (3D Matrix, Cambridge, Mass. under a material transfer agreement) was obtained as a sterile 1% (w/v) solution in water, which was mixed 1:1 with 1×10⁶ cells in 10% (w/v) sucrose (Sigma, St Louis, Mo.), 10 mM HEPES in Dulbecco's modified medium (DMEM; Gibco) immediately before use. The final concentration of cells in RAD16 hydrogel was 1×10⁶ cells/100 microliters.

Test Material (N=4/Rx)

1. Vicryl non-woven+1×10⁶ umbilicus-derived cells

2. 35/65 PCL/PGA foam+1×10⁶ umbilicus-derived cells

3. RAD 16 self-assembling peptide+1×10⁶ umbilicus-derived cells

4. Vicryl non-woven+1×10⁶ placenta-derived cells

5. 35/65 PCL/PGA foam+1×10⁶ placenta-derived cells

6. RAD 16 self-assembling peptide+1×10⁶ placenta-derived cells

7. 35/65 PCL/PGA foam

8. Vicryl non-woven

Animal Preparation. The animals were handled and maintained in accordance with the current requirements of the Animal Welfare Act. Compliance with the above Public Laws were accomplished by adhering to the Animal Welfare regulations (9 CFR) and conforming to the current standards promulgated in the Guide for the Care and Use of Laboratory Animals, 7th edition.

Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc., Indianapolis, Ind.), 5 weeks of age. All handling of the SCID mice took place under a hood. The mice were individually weighed and anesthetized with an intraperitoneal injection of a mixture of 60 milligrams/kg KETASET (ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa) and 10 milligrams/kg ROMPUN (xylazine, Mobay Corp., Shawnee, Kans.) and saline. After induction of anesthesia, the entire back of the animal from the dorsal cervical area to the dorsal lumbosacral area was clipped free of hair using electric animal clippers. The area was then scrubbed with chlorhexidine diacetate, rinsed with alcohol, dried, and painted with an aqueous iodophor solution of 1% available iodine. Ophthalmic ointment was applied to the eyes to prevent drying of the tissue during the anesthetic period.

Subcutaneous Implantation Technique. Four skin incisions, each approximately 1.0 cm in length, were made on the dorsum of the mice. Two cranial sites were located transversely over the dorsal lateral thoracic region, about 5-mm caudal to the palpated inferior edge of the scapula, with one to the left and one to the right of the vertebral column. Another two were placed transversely over the gluteal muscle area at the caudal sacro-lumbar level, about 5-mm caudal to the palpated iliac crest, with one on either side of the midline. Implants were randomly placed in these sites in accordance with the experimental design. The skin was separated from the underlying connective tissue to make a small pocket and the implant placed (or injected for RAD 16) about 1-cm caudal to the incision. The appropriate test material was implanted into the subcutaneous space. The skin incision was closed with metal clips.

Animal Housing. Mice were individually housed in microisolator cages throughout the course of the study within a temperature range of 64° F.-79° F. and relative humidity of 30% to 70%, and maintained on an approximate 12 hour light/12 hour dark cycle. The temperature and relative humidity were maintained within the stated ranges to the greatest extent possible. Diet consisted of Irradiated Pico Mouse Chow 5058 (Purina Co.) and water fed ad libitum.

Mice were euthanized at their designated intervals by carbon dioxide inhalation. The subcutaneous implantation sites with their overlying skin were excised and frozen for histology.

Histology. Excised skin with implant was fixed with 10% neutral buffered formalin (Richard-Allan Kalamazoo, Mich.). Samples with overlying and adjacent tissue were centrally bisected, paraffin-processed, and embedded on cut surface using routine methods. Five-micron tissue sections were obtained by microtome and stained with hematoxylin and eosin (Poly Scientific Bay Shore, N.Y.) using routine methods.

Results

There was minimal ingrowth of tissue into foams (without cells) implanted subcutaneously in SCID mice after 30 days. In contrast there was extensive tissue fill in foams implanted with umbilical-derived cells or placenta-derived cells. Some tissue ingrowth was observed in Vicryl non-woven scaffolds. Non-woven scaffolds seeded with umbilicus- or placenta-derived cells showed increased matrix deposition and mature blood vessels.

Summary. Synthetic absorbable non-woven/foam discs (5.0 mm diameter×1.0 mm thick) or self-assembling peptide hydrogel were seeded with either cells derived from human umbilicus or placenta and implanted subcutaneously bilaterally in the dorsal spine region of SCID mice. The results demonstrated that these cells could dramatically increase good quality tissue formation in biodegradable scaffolds.

Example 17 Use of Umbilical Cord Tissue- and Placenta-Derived Cells in Nerve Repair

Retinal ganglion cell (RGC) lesions have been extensively used as models for various repair strategies in the adult mammalian CNS. It has been demonstrated that retrobulbar section of adult rodent RGC axons results in abortive sprouting (Zeng B Y et al. J. Anat. 186:495-508 (1995)) and progressive death of the parent cell population (Villegas-Perez et al., J. Neurosci. 8:265-80 (1988)). Numerous studies have demonstrated the stimulatory effects of various exogenous and endogenous factors on the survival of axotomized RGC's and regeneration of their axons (Yip and So, Prog Retin Eye Res. 19: 559-75 (2000); Fischer D et al. Exp Neurol. 172: 257-72 (2001)). Furthermore, other studies have demonstrated that cell transplants can be used to promote regeneration of severed nerve axons (Li et al., 2003; Ramon-CuetoA. et al., Neuron 25: 425-35 (2000)). Thus, these and other studies have demonstrated that cell based therapy can be utilized for the treatment of neural disorders that affect the spinal cord, peripheral nerves, pudendal nerves, optic nerves or other diseases/trauma due to injury in which nervous damage can occur.

Self-assembling peptides (PuraMatrix™, U.S. Pat. Nos. 5,670,483, 5,955,343, US/PCT applications US2002/0160471, WO 02/062969) have been developed to act as a scaffold for cell-attachment to encapsulate cells in 3-D, plate cells in 2-D coatings, or as microcarriers in suspension cultures. Three-dimensional cell culture has required either animal-derived materials (mouse sarcoma extract), with their inherent reproducibility and cell signaling issues, or much larger synthetic scaffolds, which fail to approximate the physical nanometer-scale and chemical attributes of native ECM. RAD 16 (NH₂-(RADA)₃-COOH) and KLD (NH₂-(KLDL)₃-COOH) are synthesized in small (RAD16 is 5 nanometers) oligopeptide fragments that self-assemble into nanofibers on a scale similar to the in vivo extracellular matrix (ECM) (3D Matrix, Inc., Cambridge, Mass.). The self-assembly is initiated by mono- or di-valent cations found in culture media or the physiological environment. In the protocols described in this example, RAD 16 was used as a microcarrier for the implantation of umbilical cord tissue- and placenta-derived cells into the ocular defect. In this example, it has been demonstrated that transplants of umbilical cord tissue- and placenta-derived cells can provide efficacy in an adult rat optic nerve axonal regeneration model.

Methods & Materials

Cells. Cultures of human adult umbilicus and placenta derived cells and fibroblast cells (passage 10) were expanded for 1 passage. All cells were initially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks in Growth Medium with 100 Units per milliliter penicillin, 100 micrograms per milliliter streptomycin, 0.25 micrograms per milliliter amphotericin B (Invitrogen, Carlsbad, Calif.). At passage 11 cells were trypsinized and viability was determined using trypan blue staining Briefly, 50 microliters of cell suspension was combined with 50 microliters of 0.04% w/v trypan blue (Sigma, St. Louis Mo.) and the viable cell number, was estimated using a hemocytometer. Cells were then washed three times in supplement free-Leibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.). Cells were then suspended at a concentration of 200,000 cells in 25 microliters of RAD-16 (3DM Inc., Cambridge, Mass.) which was buffered and made isotonic as per manufacturer's recommendations. One hundred microliters of supplement free Leibovitz's L-15 medium was added above the cell/matrix suspension to keep it wet until use. These cell/matrix cultures were maintained under standard atmospheric conditions until transplantation occurred. At the point of transplantation, the excess medium was removed.

Animals and Surgery. Long Evans female rats (220-240 gram body weight) were used. Under intraperitoneal tribromoethanol anesthesia (20 milligram/100 grams body weight), the optic nerve was exposed, and the optic sheath was incised intraorbitally at approximately 2 millimeters from the optic disc, the nerve was lifted from the sheath to allow complete transsection with fine scissors (Li Y et al., 2003, J. of Neuro. 23(21):7783-7788). The completeness of transsection was confirmed by visually observing complete separation of the proximal and distal stumps. The control group consisted of lesioned rats without transplants. In transplant rats cultured postpartum cells seeded in RAD-16 were inserted between the proximal and distal stumps using a pair of microforceps. Approximately 75,000 cells in RAD-16 were implanted into the severed optic nerve. Cell/matrix was smeared into the severed cut using a pair of fine microforceps. The severed optic nerve sheath was closed with 10/0 black monofilament nylon (Ethicon, Inc., Edinburgh, UK). Thus, the gap was closed by drawing the cut proximal and distal ends of the nerve in proximity with each other.

After cell injections were performed, animals were injected with dexamethasone (2 milligrams/kilogram) for 10 days post transplantation. For the duration of the study, animals were maintained on oral cyclosporine A (210 milligrams/liter of drinking water; resulting blood concentration: 250-300 micrograms/liter) (Bedford Labs, Bedford, Ohio) from 2 days pre-transplantation until end of the study. Food and water were available ad libitum. Animals were sacrificed at either 30 or 60 days post transplantation.

CTB Application. Three days before animals were sacrificed, under anesthesia, a glass micropipette with a 30-50 millimeter tip was inserted tangentially through the sclera behind the lens, and two 4-5 microliter aliquots of a 1% retrograde tracer-cholera toxin B (CTB) aqueous solution (List Biologic, Campbell, Calif.) was injected into the vitreous. Animals were perfused with fixative and optic nerves were collected in the same fixative for 1 hour. The optic nerves were transferred into sucrose overnight. Twenty micrometer cryostat sections were incubated in 0.1 molar glycine for 30 minutes and blocked in a PBS solution containing 2.5% bovine serum albumin (BSA) (Boeringer Mannheim, Mannheim, Germany) and 0.5% triton X-100 (Sigma, St. Louis, Mo.), followed by a solution containing goat anti-CTB antibody (List Biologic, Campbell, Calif.) diluted 1:4000 in a PBS containing 2% normal rabbit serum (NRS) (Invitrogen, Carlsbad, Calif.), 2.5% BSA, and 2% Triton X-100 (Sigma, St. Louis, Mo.) in PBS, and incubated in biotinylated rabbit anti-goat IgG antibody (Vector Laboratories, Burlinghame, Calif.) diluted 1:200 in 2% Triton-X100 in PBS for 2 hours at room temperature. This was followed by staining in 1:200 streptavidin-green (Alexa Flour 438; Molecular Probes, Eugene, Oreg.) in PBS for 2 hours at room temperature. Stained sections were then washed in PBS and counterstained with propidium iodide for confocal microscopy.

Histology Preparation. Briefly, 5 days after CTB injection, rats were perfused with 4% paraformaldehyde. Rats were given 4 cubic centimeters of urethane and were then perfused with PBS (0.1 molar) then with 4% Para formaldehyde. The spinal cord was cut and the bone removed from the head to expose the colliculus. The colliculus was then removed and placed in 4% paraformaldehyde. The eye was removed by cutting around the outside of the eye and going as far back as possible. Care was given not to cut the optic nerve that lies on the underside of the eye. The eye was removed and the muscles were cut exposing the optic nerve this was then placed in 4% paraformaldehyde.

Results

Lesions alone. One month after retrotubular section of the optic nerve, a number of CTB-labeled axons were identified in the nerve segment attached to the retina. In the 200 micrometers nearest the cut, axons were seen to emit a number of collaterals at right angles to the main axis and terminate as a neuromatous tangle at the cut surface. In this cut between the proximal and distal stumps, the gap was observed to be progressively bridged by a 2-3 millimeter segment of vascularized connective tissue; however, no axons were seen to advance into this bridged area. Thus, in animals that received lesion alone no axonal growth was observed to reach the distal stump.

RAD-16 transplantation. Following transplantation of RAD-16 into the cut, visible ingrowth of vascularized connective tissue was observed. However, no axonal in growth was observed between the proximal and distal stumps. The results demonstrate that application of RAD-16 alone is not sufficient for inducing axonal regeneration in this situation.

Transplantation of umbilical cord tissue or placenta-derived cells. Transplantation of cells into the severed optic nerve stimulated optic nerve regrowth. Some regrowth was also observed in conditions in which fibroblast cells were implanted, although this was minimal as compared with the regrowth observed with the transplanted placenta-derived cells. Optic nerve regrowth was observed in 4/5 animals transplanted with placenta-derived cells, 3/6 animals transplanted with adult dermal fibroblasts and in 1/4 animals transplanted with umbilicus-derived cells. In situations where regrowth was observed, CTB labeling confirmed regeneration of retinal ganglion cell axons, which were demonstrated to penetrate through the transplant area. GFAP labeling was also performed to determine the level of glial scarring. The GFAP expression was intensified at the proximal stump with some immunostaining being observed through the reinervated graft.

Summary. These results demonstrate that transplanted human adult placenta and umbilical cord tissue-derived cells are able to stimulate and guide regeneration of cut retinal ganglion cell axons.

Example 18 Effect of Treatment with Umbilical-Derived Cells on Transient Middle Cerebral Artery Occlusion Stroke (tMCAo)

Umbilical-derived cells were evaluated in rats subjected to two-hour middle cerebral artery occlusion (MCAo). Behavioral tests for functional recovery were assessed at various time points and compared with vehicle and mesenchymal stem cell controls. Histology was performed at sacrifice to evaluate improvements and the tissue and cellular level.

Materials and Methods

Cell preparation and transplantation. Postpartum umbilicus-derived cells and human mesenchymal stem cells (Cambrex, PT2501) were cultured in-house before shipping to the site of the study, where they were thawed and processed on the day of transplantation. Cells were thawed in a 37° C. water bath, washed with 10 mL PBS in a 15 mL tube and centrifuged at 1000 rpm for 5 minutes at 4° C. The supernatant was discarded and cells resuspended in PBS. Cells were kept on ice prior to implantation, and viability determined by Trypan blue staining In this study cell viability averaged 80% for PPDCs and 92% for MSCs.

At 1 day post-ischemia, randomly selected animals underwent cell transplantation. Animals were anesthetized with 3.5% halothane in N₂O:O₂ (2:1) and maintained at 0.5% halothane via a facemask. Each experimental group received cells suspended in 2 ml PBS or PBS alone administered via tail vein injection (27G1/2, B-D Precision Glide). The four groups (N=8/group) were 1 million umbilical-derived cells (U1), 3 million umbilical-derived cells (U3), 3 million MSCs (MSC), and PBS vehicle control. Immunosuppressants were not applied.

Temporal Middle Cerebral Artery Occlusion. Middle cerebral artery occlusion (MCAo) was induced in two- to three-month old Wistar rats (male, 270 to 300 g) using a modified method of intraluminal vascular occlusion (Chen et al., 1992). Briefly, rats were anesthetized with 3.5% halothane in N2O:O2 (2:1) and maintained at 0.5% halothane via a facemask. The right common carotid artery, external carotid artery and internal carotid artery were exposed. A length of 3-0 monofilament nylon suture, varying between 18.5 to 19.5 mm, depending on the animal weight, with its tip rounded by heating near a flame, was advanced from the external carotid artery into the lumen of the internal carotid artery until it blocked the origin of the MCA. Two hours after MCAo, reperfusion was performed by withdrawal of the suture until the tip cleared the internal carotid artery.

Neurological functional tests. Four behavioral tests were performed before MCAo (baseline), one day after MCAo, and weekly thereafter by an investigator who was blinded to the experimental groups.

Modified neurological severity score (mNSS) is a composite of motor, sensory, balance and reflex tests (Chen et al., 2001). Neurological function was graded on a scale of 0 to 18 (normal score 0; maximal deficit score 18) with one point awarded for the exhibition of specific abnormal behavior or for lack of a tested reflex. A greater impairment of normal function resulted in a higher score.

In the foot-fault test, rats were tested for placement dysfunctions of forelimb (Zhang et al., 2002). Rats were placed on elevated hexagonal grids of different sizes. The total number of steps used to cross the grid was counted, and the total number of foot faults for each forelimb was recorded. It is well established that in animals with MCAo, the impaired (contralateral) limbs faulted more often than non-impaired limbs.

The adhesive removal patch test requires the use of adhesive-backed paper dots of equal size (113.1 mm²) as bilateral tactile stimuli on the distal-radial region of each forelimb (Schallert et al., 1997). Prior to surgery rats were trained for 3 days to remove the adhesive. Once the rats were able to remove the adhesive dots within 10 seconds, they were subjected to MCAo. Post-MCAo, the time to remove each stimulus from both forelimbs was recorded on 3 trials per day. Individual trials were separated by at least 5 min.

The corner test measures sensory and motor functions and is more sensitive for long-term sensory and motor deficits (Zhang et al., 2002). A rat was placed between two boards (dimensions of 30×20×1 cm³) attached at 30° with a small opening along the joint to encourage entry into the corner. The rat was placed facing the corner such that both sides of the vibrissae were stimulated together when it entered deep into the corner. The rat would rear forward and upward, then turn back to face the open end. Healthy uninjured rats would turn either left or right; in contrast MCAo-injured rats preferentially turned toward the non-impaired side. The number of turns in one versus the other direction was recorded from ten trials for each test, and the percentage of turns used as the corner test score.

Brain section preparation. To identify cell proliferation, all rats received intraperitoneal injections of bromodeoxyuridine (BrdU, 50 mg/kg, Sigma, St, Louis Mo.) daily for 14 consecutive days after MCAo. Rat brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde. Using a rat brain matrix (Activational Systems Inc., Warren, Mich.), each forebrain was cut into 2-mm thick coronal blocks for a total of 7 blocks from bregma 5.2 mm to bregma −8.8 mm per animal (Paxinos and Watson 1986 The rat brain in stereotaxic coordinated, 2^(nd) Ed. Sydney; Orlando. Academic Press). The tissues were embedded in paraffin, and a series of 6 μm-thick slides was cut.

Measurement of infarct area. One of each coronal paraffin slides (6 μm thick) from seven blocks was stained with hematoxylin and eosin (H&E). Seven brain slides were traced using the Global Lab Image analysis system (Data Translation, Marlboro, Mass.). The indirect lesion area, in which the intact area of the ipsilateral hemisphere was subtracted from the area of the contralateral hemisphere, was calculated (Swanson et al., 1990). The lesion volume is presented as a volume percentage of the lesion compared to the contralateral hemisphere.

Immunohistochemical Assessment. A standard paraffin block was obtained from the center of the lesion, corresponding to coronal coordinates for bregma-1˜1 mm. A series of 6-μm-thick sections were cut from this block for analysis by light and fluorescent microscopy (Olympus, BH-2). To identify grafted human cells, a mouse anti-human nuclear monoclonal antibody (mAb 1281, Chemicon International, Inc) was used at a titer of 1:300. To distinguish proliferating cells, a mouse antibody (Dako) was applied at a titer of 1:100. Double staining was performed to visualize cellular co-localization of human nuclei or BrdU with cell type-specific markers (NeuN for neurons, dilution 1:500; glial fibrillary acidic protein, GFAP, for astrocytes, dilution 1:5000). Moreover, antibodies against von Willebrand factor (vWF, 1:400, Dako) and synaptophysin (1:500, Chemicon) were used to identify vessels and synapses, respectively.

Apoptotic cell staining. The terminal deoxynucleotidyl transferase (TdT)—mediated dUTP-biotin nick end labeling (TUNEL) method (in situ Apoptosis Detection Kit, Chemicon) was used to assess in situ apoptotic detection (Li et al., 1998, Li et al., 1995). The TUNEL method is based on the specific binding of TdT to 3′-OH ends of DNA and the ensuing synthesis of polydeoxynucleotide polymer cells. Staining was performed according to the manufacturer's protocol.

Cell Quantification. To quantify donor human cells and apoptotic cells, the number of apoptotic cells in ipsilateral hemisphere per section was counted. For measurement of vascular and synapse density, 8 fields of view from the ischemic penumbra were digitized using either a 40× objective via the MCID computer imaging analysis system. The number of vessels as well as the positive area of synaptophysin in the ischemic boundary zone (IBZ) were counted throughout each field of view.

Results

Neurological Functional Outcome. Severe behavioral deficits were evident in all animals 1 day post-stroke. All rats subjected to MCAo showed progressive behavioral recovery over time to 28 days post-MCAo (see Table 18-1). No significant differences in behavioral tests were detected amongst groups before MCAo in all the functional tests. At 28 days post-MCAo, mNSS injury had progressed from moderate to mild: statistically significant improvement was observed in mNSS in cell-treated groups compared to PBS control (p=0.01 for U1, p=0.1 for U3, and p=0.02 for MSC). The percentage of recorded foot faults also decreased in a statistically significant manner in rats receiving cell therapies (p=0.02 for U1, p=0.06 for U3, and p=0.01 for MSC).

No significant differences were observed in the adhesive removal as well as corner tests. The weight of animals in all groups showed a similar trend of loss immediately after MCAo followed by steady gain within the first week.

TABLE 18-1 Neurological functional outcome PBS MSC U1 U3 Time (days) Mean S.E. Mean S.E. Mean S.E. Mean S.E. mNSS  2 11.25 0.31 11.00 0.57 10.50 0.19 10.63 0.46  7 8.29 0.36 7.63 0.50 7.75 0.16 7.63 0.42 14 7.14 0.34 6.50 0.33 6.75 0.16 6.63 0.42 21 5.86 0.26 5.50 0.33 5.63 0.18 5.63 0.42 28 5.71 0.29 4.50 0.33 4.50 0.27 4.75 0.45 P-value (vs. PBS) 0.02 0.01 0.10 Footfault  2 38.38 1.66 37.00 2.39 36.50 1.18 37.13 1.68  7 31.43 1.99 28.88 1.92 31.13 1.48 29.50 1.99 14 25.00 1.86 20.63 1.41 25.25 1.37 22.63 1.57 21 20.00 1.23 15.63 1.27 19.13 1.44 16.63 1.43 28 16.14 1.30 11.50 0.80 11.50 1.02 12.63 1.05 P-value (vs. PBS) 0.01 0.02 0.06 Corner  2 9.38 0.42 9.88 0.13 9.63 0.26 9.50 0.33  7 9.29 0.36 9.13 0.30 9.00 0.42 9.25 0.49 14 8.86 0.55 8.50 0.33 8.13 0.35 9.13 0.58 21 7.86 0.59 8.00 0.27 7.38 0.32 8.50 0.60 28 7.43 0.61 7.50 0.33 6.63 0.38 7.75 0.65 P-value (vs. PBS) 0.92 0.29 0.72 Adhesive  2 108.38 7.78 110.63 6.97 103.50 8.49 107.88 7.98  7 89.14 15.19 102.63 12.02 69.25 10.57 75.50 14.07 14 75.43 15.15 79.88 12.76 54.38 10.71 70.50 12.95 21 64.43 15.76 69.13 11.64 43.63 9.37 52.88 13.12 28 38.29 9.76 45.50 9.36 32.75 7.30 45.63 13.50 P-value (vs. PBS) 0.60 0.66 0.67 Weight  0 296.38 4.71 296.50 6.20 292.00 4.50 300.75 6.05  2 259.63 6.60 258.63 8.93 258.88 4.07 261.88 9.30  7 272.14 13.77 283.00 15.42 281.75 8.00 264.63 18.68 14 311.57 11.15 319.13 14.02 315.50 8.50 292.75 22.33 21 348.00 12.54 356.00 13.24 344.00 11.47 316.38 26.13 28 376.86 10.96 384.75 12.97 373.25 13.91 342.75 28.94 P-value (vs. PBS) 0.65 0.84 0.30

Infarct volume. As shown in Table 18-2, no significant differences of the percent lesion volumes to the contralateral hemisphere were detected among all four groups.

TABLE 18-2 Histology results. PBS MSC U1 U3 Lesion Volume Mean 0.35 0.35 0.32 0.33 S.E. 0.04 0.04 0.05 0.07 Synapotphysin Mean 0.19 0.26 0.22 0.23 S.E. 0.02 0.02 0.02 0.02 Number of vWF positive vessels/40x field Mean 15.54 18.24 19.35 18.82 S.E. 0.57 0.58 0.54 0.60 vWF positive vessels density per 40x field Mean 2.76 3.75 3.15 2.97 S.E. 0.29 0.25 0.24 0.32 Apoptosis Mean 90.86 55.38 44.88 56.38 S.E. 12.29 8.01 6.58 5.59 BrdU/Contralateral Mean 21.29 24.13 20.75 23.50 S.E. 2.18 1.89 2.80 3.45 BrdU/Ipsilateral Mean 26.57 43.13 28.50 43.88 S.E. 3.12 5.44 4.28 8.22 MAB1281 Mean 73.86 45.50 58.13 S.E. 7.36 8.57 7.78

Histology. Donor cells in the brain. Within the brain tissue, human cells were characterized by round-to-oval nuclei identified by the human specific antibody mAb1281. Donor cells survived and were distributed throughout the ischemic damaged brain of recipient rats. Double immunohistochemical staining of brain sections revealed approximately 1% of human mAb1281-positive cells to be reactive for the neuronal marker, NeuN, and for the astrocytic marker, GFAP (Table 18-2).

Neurogenesis in the ischemic brain. BrdU, a thymidine analog, can be incorporated into cells during S phase, and BrdU-positive cells in the subventricular zone (SVZ) are considered to be progenitor cells undergoing proliferation. In this study, rats treated with higher doses of cells showed an increase in proliferation in the SVZ in the ipsilateral hemisphere. Progenitor cells in the SVZ can remain in either a proliferating state or they can exit the cell cycle and differentiate into functional brain cells. Since the latter property of neural progenitor cells is tied in with brain self-repair after injury or disease, double staining for BrdU and GFAP or NeuN was performed and showed approximately 5% BrdU-positive cells expressed GFAP or NeuN (Table 18-2).

Angiogenesis (vascular modulation). The presence of enlarged, thin-walled vessels in the ischemic boundary zone (IBZ) is indicative of angiogenesis (Zhang et al. (2002) J. Cereb. Blood Flow Metab. 22:379-92). Cell treatments were observed to increase the number of vessels by 17 to 25% in the IBZ compared to PBS vehicle control (Table 18-2).

Synaptogenesis. Synaptophysin, a pre-synaptic vesicle protein, is used as an indicator of synaptogenesis (Ujike et al. (2002) Ann. Ny Acad. Sci. 965:55-67). In this study, an increase in synaptophysin expression in the IBZ was observed with cell treatments (Table 18-2).

Reduction of cell apoptosis and death. TUNEL staining showed apoptotic cells with typical dark brown, rounded or oval apoptotic bodies were present throughout the damaged tissue, although the vast majority of apoptosis was located in the IBZ or SVZ. The number of apoptotic cells in the ipsilateral hemisphere was reduced by 51% in rats receiving U1 cells and by 38% in U3- and MSC-treated rats (P<0.01 for U1, P<0.05 for MSC and U3, compared to PBS control) (Table 18-2).

Summary

PPDCs were demonstrated to have utility for the treatment of strokes. Cell therapy led to improved neurological functions evidenced through higher motor-sensory scores. PPDCs administered into the tail vein after MCAo entered the ischemic brain tissue and survived at least 28 days after cell injection without immunosuppressants. The positive effects became statistically significant at 21 days post-MCAo, persisting to the last day of observation (28 days post-surgery). Significantly more microvessels existed in the ipsilateral hemisphere of rats treated with PPDC cells (compared to control animal, P<0.05). PPDC treatment also significantly reduced the number of apoptotic cells in the ipsilateral hemisphere at 28 days after MCAo.

References for Example 18

-   1. Chen, H., M. Chopp, Z. G. Zhang, and J. H. Garcia. 1992. J Cereb     Blood Flow Metab 12:621-8. -   2. Chen, J., Y. Li, L. Wang, Z. Zhang, D. Lu, M. Lu, and M.     Chopp. 2001. Stroke 32:1005-11. -   3. Guide for the Care and Use of Laboratory Animals, 7th edition. -   4. Li, Y., C. Powers, N. Jiang, and M. Chopp. 1998. J Neurol Sci     156:119-32. -   5. Li, Y., V. G. Sharov, N. Jiang, C. Zaloga, H. N. Sabbah, and M.     Chopp. 1995. Am J Pathol 146:1045-51. -   6. Paxinos, G., and C. Watson. 1986. The rat brain in stereotaxic     coordinates, 2nd edition. Sydney; Orlando: Academic Press. -   7. Schallert, T., D. A. Kozlowski, J. L. Humm, and R. R.     Cocke. 1997. Adv Neurol 73:229-38. -   8. Swanson, R. A., M. T. Morton, G. Tsao-Wu, R. A. Savalos, C.     Davidson, and F. R. Sharp. 1990. J Cereb Blood Flow Metab 10:290-3. -   9. Trenka-Benthin, S. (2005). Testicular lesion histology report and     photomicrograph indexes. -   10. Ujike, H., M. Takaki, M. Kodama, and S. Kuroda. 2002 Ann N Y     Acad Sci 965:55-67. -   11. Zhang, L., T. Schallert, Z. G. Zhang, Q. Jiang, P. Arniego, Q.     Li, M. Lu, and M. Chopp. 2002a. J Neurosci Methods 117:207-14. -   12. Zhang, Z. G., L. Zhang, W. Tsang, H. Soltanian-Zadeh, D.     Morris, R. Zhang, A. Goussev, C. Powers, T. Yeich, and M. Chopp.     2002b. J Cereb Blood Flow Metab 22:379-92.

Example 19 Endothelial Network Formation Assay

Angiogenesis, or the formation of new vasculature, is necessary for the growth of new tissue. Induction of angiogenesis is an important therapeutic goal in many pathological conditions. The present study was aimed at identifying potential angiogenic activity of the umbilical-derived cells in in vitro assays. The study followed a well-established method of seeding endothelial cells onto a culture plate coated with MATRIGEL™ (BD Discovery Labware, Bedford, Mass.), a basement membrane extract (Nicosia and Ottinetti (1990) In vitro Cell Dev. Biol. 26(2):119-28). Treating endothelial cells on MATRIGEL (BD Discovery Labware, Bedford, Mass.) with angiogenic factors will stimulate the cells to form a network that is similar to capillaries. This is a common in vitro assay for testing stimulators and inhibitors of blood vessel formation (Ito et al. (1996) Int. J. Cancer 67(1):148-52). The present studies made use of a co-culture system with the postpartum-derived cells seeded onto culture well inserts. These permeable inserts allow for the passive exchange of media components between the endothelial and the umbilical-derived cell culture media.

Methods & Materials

Cell Culture.

Placenta and Umbilical Cord-derived cells. Human umbilical cords and placenta were received and cells were isolated as previously described (Example 1). Cells were cultured in Growth medium (Dulbecco's Modified Essential Media (DMEM; Invitrogen, Carlsbad, Calif.), 15% (v/v) fetal bovine serum (Hyclone, Logan Utah), 100 Units/milliliter penicillin, 100 microgram/milliliter streptomycin (Invitrogen), 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.)) on gelatin-coated tissue culture plastic flasks. The cultures were incubated at 37° C. with 5% CO₂. Cells used for experiments were between passages 4 and 12.

Actively growing placenta and umbilical cord-derived cells were trypsinized, counted, and seeded onto COSTAR TRANSWELL 6.5 millimeter diameter tissue culture inserts (Corning, Corning, N.Y.) at 15,000 cells per insert. Cells were cultured on the inserts for 48-72 hours in Growth medium at 37° C. under standard growth conditions.

Human mesenchymal stem cells (hMSC). hMSCs were purchased from Cambrex (Walkersville, Md.) and cultured in MSCGM (Cambrex). The cultures were incubated under standard growth conditions.

Actively growing MSCs were trypsinized and counted and seeded onto COSTAR TRANSWELL 6.5 millimeter diameter tissue culture inserts (Corning, Corning, N.Y.) at 15,000 cells per insert. Cells were cultured on the inserts for 48 to 72 hours in Growth medium under standard growth conditions.

Human umbilical vein endothelial cells (HUVEC). HUVEC were obtained from Cambrex (Walkersville, Md.). Cells were grown in separate cultures in either EBM or EGM endothelial cell media (Cambrex). Cells were grown on standard tissue-cultured plastic under standard growth conditions. Cells used in the assay were between passages 4 and 10.

Human coronary artery endothelial cells (HCAEC). HCAEC were purchased from Cambrex Incorporated (Walkersville, Md.). These cells were also maintained in separate cultures in either the EBM or EGM media formulations. Cells were grown on standard tissue cultured plastic under standard growth conditions. Cells used for experiments were between passages 4 and 8.

Endothelial Network Formation (MATRIGEL™) assays. Culture plates were coated with MATRIGEL™ (BD Discovery Labware, Bedford, Mass.) according to manufacturer's specifications. Briefly, MATRIGEL™ (BD Discovery Labware, Bedford, Mass.) was thawed at 4° C. and approximately 250 microliters were aliquoted and distributed evenly onto each well of a chilled 24-well culture plate (Corning). The plate was then incubated at 37° C. for 30 minutes to allow the material to solidify. Actively growing endothelial cell cultures were trypsinized and counted. Cells were washed twice in Growth medium with 2% FBS by centrifugation, resuspension, and aspiration of the supernatant. Cells were seeded onto the coated wells at 20,000 cells per well in approximately 0.5 milliliter Growth medium with 2% (v/v) FBS. Cells were then incubated for approximately 30 minutes to allow cells to settle.

Endothelial cell cultures were then treated with either 10 nanomolar human bFGF (Peprotech, Rocky Hill, N.J.) or 10 nanomolar human VEGF (Peprotech, Rocky Hill, N.J.) to serve as a positive control for endothelial cell response. Transwell inserts seeded with placental or umbilical-derived cells were added to appropriate wells with Growth medium with 2% FBS in the insert chamber. Cultures were incubated at 37° C. with 5% CO₂ for approximately 24 hours. The well plate was removed from the incubator, and images of the endothelial cell cultures were collected with an Olympus inverted microscope (Olympus, Melville, N.Y.).

Results

In a co-culture system with placenta-derived cells or with umbilical cord-derived cells, HUVEC form cell networks (data not shown). HUVEC cells form limited cell networks in co-culture experiments with hMSCs and with 10 nanomolar bFGF (data not shown). HUVEC cells without any treatment showed very little or no network formation (data not shown). These results suggest that the umbilical-derived cells release angiogenic factors that stimulate the HUVEC.

In a co-culture system with placenta-derived cells or with umbilical cord-derived cells, CAECs form cell networks (data not shown).

Table 19-1 shows levels of known angiogenic factors released by the umbilical- and placental-derived cells in Growth medium. Postpartum-derived cells were seeded onto inserts as described above. The cells were cultured at 37° C. in atmospheric oxygen for 48 hours on the inserts and then switched to a 2% FBS media and returned at 37° C. for 24 hours. Media was removed, immediately frozen and stored at −80° C., and analyzed by the SearchLight multiplex ELISA assay (Pierce Chemical Company, Rockford, Ill.). Results shown are the averages of duplicate measurements. The results show that the do not release detectable levels of platelet-derived growth factor-bb (PDGF-bb) or heparin-binding epidermal growth factor (HBEGF). The cells release measurable quantities of tissue inhibitor of metallinoprotease-1 (TIMP-1), angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF).

TABLE 19-1 Potential angiogenic factors released from umbilical and placental-derived cells. TIMP1 ANG2 PDGFBB TPO KGF HGF FGF VEGF HBEGF (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) Plac 91655.3 175.5 <2.0 275.5 3.0 58.3 7.5 644.6 <1.2 (P4) Plac 1592832.4 28.1 <2.0 1273.1 193.3 5960.3 34.8 12361.1 1.7 (P11) Umb 81831.7 <9.8 <2.0 365.9 14.1 200.2 5.8 <4.0 <1.2 cord (P4) Media <9.8 25.1 <2.0 <6.4 <2.0 <3.2 <5.4 <4.0 <1.2 alone Plac: placenta derived cells; Umb cord: Umbilical cord derived cells Umbilical and placental-derived cells were cultured in 24 hours in media with 2% FBS in atmospheric oxygen. Media was removed and assayed by the SearchLight multiplex ELISA assay (Pierce). Results are the means of a duplicate analysis. Values are concentrations in the media reported in picograms per milliliter of culture media.

Table 19-2 shows levels of known angiogenic factors released by the umbilical and placental-derived cells. The cells were seeded onto inserts as described above. The cells were cultured in Growth medium at 5% oxygen for 48 hours on the inserts, then switched to a 2% FBS medium, and returned to 5% O₂ incubation for 24 hours. Media was removed, immediately frozen, stored at −80° C., and analyzed by the SearchLight multiplex ELISA assay (Pierce Chemical Company, Rockford, Ill.). Results shown are the averages of duplicate measurements. The results show that the umbilical- and placental-derived cells do not release detectable levels of platelet-derived growth factor-bb (PDGF-BB) or heparin-binding epidermal growth factor (HBEGF). The cells do release measurable quantities of tissue inhibitor of metallinoprotease-1 (TIMP-1), angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF).

TABLE 19-2 Potential angiogenic factors released from umbilical and placental-derived cells. PDGF- HB- TIMP1 ANG2 BB TPO KGF HGF FGF VEGF EGF (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) Plac 72972.5 253.6 <2.0 743.1 2.5 30.2 15.1 1495.1 <1.2 (P4) Plac 458023.1 55.1 <2.0 2562.2 114.2 2138.0 295.1 7521.3 1.8 (P11) Umb 50244.7 <9.8 <2.0 403.3 10.7 156.8 5.7 <4.0 <1.2 cord (P4) Media <9.8 25.1 <2.0 <6.4 <2.0 <3.2 <5.4 <4.0 <1.2 alone Plac: placenta derived cells; Umb cord: Umbilical cord derived cells Umbilical and placental-derived cells were cultured in 24 hours in media with 2% FBS in 5% oxygen. Media was removed and assayed by the SearchLight multiplex ELISA assay (Pierce). Results are the means of a duplicate analysis. Values are concentrations in the media reported in picograms per milliliter of culture media.

Summary. The results of the present study show that umbilical-derived cells can stimulate both human umbilical vein and coronary artery endothelial cells to form networks in an in vitro MATRIGEL™ (BD Discovery Labware, Bedford, Mass.) assay. This effect is similar to that seen with known angiogenic factors in this assay system. These results suggest that the umbilical-derived cells are useful for stimulating angiogenesis in vivo.

Example 20 RayBio® and BD Powerblot™ Cytokine Arrays

RayBio® Human Cytokine Antibody Array C Series 1000 was used to analyze the expression of 120 proteins in umbilical-derived cells and lysates. This analysis provided a characterization of the UTCs and identified an expression spectrum of key trophic factors for these cells.

Materials and Methods

Cell Growth and Harvest. Umbilical-derived cells were seeded at 5,000 cells per cm² in gelatin-coated flasks with growth media and expanded for 3 to 4 days (25,000 cells per cm² target harvest density). Cells were harvested with trypsin, collected, and centrifuged at 300 rcf for 5 minutes. The trypsin/media was removed by aspiration and cells were washed three times with phosphate buffered saline (PBS).

Cell Wash and Aliquoting. After washing, the cells were re-suspended at 10⁷ cell/ml in PBS and delivered as 1 ml aliquots into 1.5 ml sterile siliconized micro-centrifuge tubes. The cells were centrifuged at 300 rcf for 5 minutes and the PBS was removed by aspiration. Cells were either lyzed and analyzed by the array, or lyzed and lyophilized for analysis.

Preparation of Lyophilized Samples. Three lots of cells (UTC Lots L040405, L052505, L050505) were prepped for eventual lyophilization by immersing into liquid nitrogen (LN2) for 60 seconds. The tubes were then removed from LN2 and immediately immersed in a 37° C. water bath for 60 seconds or until thawed (3 minute maximum incubation time). This process was repeated two additional times. The freeze-thawed samples were centrifuged for 10 minutes at 13,000 rcf at 4° C. and placed on ice. The supernatant fluid from each tube was removed. To determine total protein content, lysate was diluted into PBS and the dilution was analyzed by Bradford assay.

For lyophilization, multiple 1.5 ml sterile cryovials labeled with lysate were loaded into an autoclaved and cooled heat transfer block. Aliquots of lysate supernatant fluid at defined total protein concentration were loaded into the cryovials. The heat block containing uncapped cryovials were aseptically loaded into autoclaved un-used autoclave pouch. The pouch was loaded into the lyophilizer.

Test materials with applied lysate were loaded into a FTS Systems Dura-Stop MP Stoppering Tray Dryer and lyophilized using the following ramping program. All steps had a ramping rate of 2.5° C./minute and a 100-mT vacuum.

Step Shelf Temperature (° C.) Hold Time (minutes) a −40 180 b −25 2160 c −15 180 d −5 180 e 5 120 f 20 120 g −20 60

Preparation of Cell Pellets. Frozen cell pellets (lots 063004B, 022803, 050604B, 072804, 120304, 071404C, 090304) were lysed using a 1:1 mix of RIPA buffer (50 mM Tris HCl, pH8, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate and 0.1% SDS) and cell lysis buffer provided in the RayBio® cytokine array 1000.1 kit (Raybiotech Inc. Norcross, Ga.). Glass beads (Sigma, Mo.) were used to achieve complete cell lysis. Protein concentration was measured using the BCA protein assay kit (Pierce Biotechnology, Inc. Rockford, Ill.).

RayBio® Array Analysis. RayBio® arrays VI and VII, which constitute the array 1000.1, were probed overnight with equal amounts of protein from each sample. The remaining protocol was followed as per the manufacturer's guidelines. The spots on the membrane were qualitatively analyzed to determine proteins of interest. For quantitative comparison between samples, these spots could be analyzed by densitometry and changes in expression confirmed by ELISA.

Results

A total of ten different UTC populations were analyzed. Forty-eight proteins were qualitatively identified and listed in Table 19-1. Some proteins were expressed at relatively high concentrations in all samples tested while others were expressed in certain samples.

TABLE 20-1 Qualitatively identified UTC proteins. Num- ber Trophic Factor Abbreviation 1 Brain Derived Neurotrophic Factor BDNF 2 Basic Fibroblast Growth Factor bFGF 3 Bone Morphogenetic Protein-4 BMP-4 4 Bone Morphogenetic Protein-6 BMP-6 5 MPIF-1 CK b 8-1 6 Ciliary Neurotrophic Factor CNTF 7 CCL27 (cutaneous T cell attracting chemokine) CTACK 8 Epidermal Growth Factor EGF 9 CCL26 Eotaxin-3 10 Fas Antigen Fas/ TNFRSF6 11 Fibroblast Growth Factor-6 FGF-6 12 FMS related Tyrosine Kinase 3 FIT-3 ligand 13 CS3C chemokine Fractalkine 14 Granulocyte Colony Stimulating Factor GCSF 15 Glucocorticoid Induced TNF Receptor GITR ligand Superfamily-Related Protein 16 Granulocyte-Macrophage Colony Stimulating GM-CSF Factor 17 Hepatocyte Growth Factor HGF 18 CCL1 I-309 19 Intercellular Adhesion Molecules 1 ICAM-1 20 Insulin Like Growth Factor Binding Protein-1 IGFBP-1 21 Insulin Like Growth Factor Binding Protein-2 IGFBP-2 22 Insulin Like Growth Factor Binding Protein-3 IGFBP-3 23 Insulin Like Growth Factor Binding Protein-6 IGFBP-6 24 Interleukin-10 IL-10 25 Interleukin-13 IL-13 26 Interleukin-1a IL-1a 27 Interleukin-1Ra IL-1Ra 28 Interleukin-3 IL-3 29 Interleukin-5 IL-5 30 Interleukin-6 IL-6 31 Interleukin-7 IL-7 32 Interleukin-8 IL-8 33 IFN-Inducible T Cell Chemoattractant I-TAC 34 Monocyte Chemotactic Protein-1 MCP-1 35 Migration Inhibitory Factor MIF 36 Macrophage Inflammatory Protein-1 MIP-1a 37 Oncostatin M Oncostatin M 38 Phosphatidylinositol Glycan F PIGF 39 Soluble gp130-signal transducer chain sgp130 40 Transforming Growth Factor-B1 TGF-b1 41 Transforming Growth Factor-B3 TGF-b3 42 Thrombopoietin Thrombo- poietin 43 Tissue Inhibitor of Metalloproteinase 2 TIMP-2 44 Tumor Necrosis Factor-alpha TNF-a 45 Tumor Necrosis Factor-beta TNF-b 46 TNF-Related Apoptosis-Inducing Ligand TRAIL-R3 Receptor-3 47 TNF-Related Apoptosis-Inducing Ligand TRAIL-R4 Receptor-4 48 Urokinase-Type Plasminogen Activator Receptor uPAR

Summary

The RayBio® array confirms the expression of proteins previously identified by gene array and/or ELISA analyses. Various trophic factors beneficial for specific disease treatment have been identified. For instance, FGF, TGF-b, and GCSF were identified in UTCs, and these growth factors have been previously identified with improvements in animal models of acute stroke and stroke recovery. In addition, BDNF, BMP-4, BMP-6, and TGF-b1, which are positively associated with Parkinson's disease, have been identified in UTCs. All data presented are qualitatively assessed; quantitative analysis of the level of expression for proteins of interest is pending.

Example 21 Effect of Variant PPDC Doses in Transient Middle Cerebral Artery Occlusion Stroke

hUTC cells were evaluated at different doses in rats subjected to 2-hour middle cerebral artery occlusion (MCAo). PPC treatments at varying doses: 0.3, 1, 3, and 10 million cells/dose-rat, were compared to PBS and mesenchymal stem cell controls in a 60-day study.

Materials and Methods

Cell Preparation. hUTC cells and human mesenchymal stem cells (Cambrex, PT2501) were cultured in-house before shipping to the site of study, where they were thawed and processed on the day of transplantation. Cells were thawed in a 37° C. water bath, washed with 10 ml, PBS in a 15 mL tube and centrifuged at 1000 rpm for 5 minutes at 4° C. The supernatant was discarded and cells resuspended in PBS. Cells were kept on ice prior to implantation, and viability determined by Trypan blue staining Cell viability averaged 85% for PPDC and MSCs.

At 1 day post-ischemia, randomly selected animals underwent cell transplantation. Animals were anesthetized with 3.5% halothane in N₂O:O₂ (2:1) and maintained at 0.5% halothane via a facemask. Each experimental group received cells suspended in 2 ml PBS or PBS alone administered via tail vein injection (27G1/2, B-D Precision Glide). The six groups (N=8/group) were 0.3, 1, 3, and 10 million PPCs, 3 million MSCs (“MSC”), and PBS vehicle control. Immuno-suppressants were not applied.

Temporal Middle Cerebral Artery Occlusion. Middle cerebral artery occlusion (MCAo) was induced in rats using a modified method of intraluminal vascular occlusion. In brief, rats were anesthetized with 3.5% halothane in N₂O:O₂ (2:1) and maintained at 0.5% halothane via a facemask. The right common carotid artery, external carotid artery and internal carotid artery were exposed. A length of 3-0 monofilament nylon suture, varying between 18.5 to 19.5 mm depending on the animal weight, with its tip rounded by heating near a flame, was advanced from the external carotid artery into the lumen of the internal carotid artery until it blocked the origin of the MCA. Two hours after MCAo, reperfusion was performed by withdrawal of the suture until the tip cleared the internal carotid artery.

Neurological functional tests. Four behavioral tests were performed before MCAo (baseline), one day after MCAo, and on Days 7, 14, 21, 28, 35, 42, 56, and 60 by an investigator who was blinded to the experimental groups. The details of each test were as described in EXAMPLE 18.

Results

Neurological Functional Outcome. Severe behavioral deficits were evident in all animals 1 day post-stroke. All rats subjected to MCAo showed progressive behavioral recovery over time to 60 days post-MCAo. Animals receiving PPDC treatments showed a trend towards functional recovery in all tests. Overall, the beneficial effects of cell therapy was sustained and increased over the duration of study.

A dose response was observed in mNSS (FIG. 1), foot-fault (FIG. 2), and adhesive removal (FIG. 3) tests, whereby a dose of either 3 million or 10 million PPDC led to faster, greater recovery. In the corner test (FIG. 4), PPDC-treated rats also performed better than PBS vehicle control while MSC-treated rats were worse than PBS control.

Summary

Umbilical cord-derived cells demonstrated efficacy in the treatment of stroke in the rodent MCAo model. A dose response was observed, up to the two highest UTC doses that showed similar functional improvement outcomes. Cell-treated animals continued to improve steadily over the duration of 60 days.

References for Example 21

-   1. Chen, H., M. Chopp, Z. G. Zhang, and J. H. Garcia. 1992. The     effect of hypothermia on transient middle cerebral artery occlusion     in the rat. J Cereb Blood Flow Metab 12:621-8. -   2. Chen, J., Y. Li, L. Wang, Z. Zhang, D. Lu, M. Lu, and M.     Chopp. 2001. Therapeutic benefit of intravenous administration of     bone marrow stromal cells after cerebral ischemia in rats. Stroke     32:1005-11. -   3. Guide for the Care and Use of Laboratory Animals, 7th edition. -   4. Li, Y., C. Powers, N. Jiang, and M. Chopp. 1998. Intact, injured,     necrotic and apoptotic cells after focal cerebral ischemia in the     rat. J Neurol Sci 156:119-32. -   5. Li, Y., V. G. Sharov, N. Jiang, C. Zaloga, H. N. Sabbah, and M.     Chopp. 1995. Ultrastructural and light microscopic evidence of     apoptosis after middle cerebral artery occlusion in the rat. Am J     Pathol 146:1045-51. -   6. Paxinos, G., and C. Watson. 1986. The rat brain in stereotaxic     coordinates, 2nd edition. Sydney; Orlando: Academic Press. -   7. Schallert, T., D. A. Kozlowski, J. L. Humm, and R. R.     Cocke. 1997. Use-dependent structural events in recovery of     function. Adv Neurol 73:229-38. -   8. Swanson, R. A., M. T. Morton, G. Tsao-Wu, R. A. Savalos, C.     Davidson, and F. R. Sharp. 1990. A semi-automated method for     measuring brain infarct volume. J Cereb Blood Flow Metab 10:290-3. -   9. Ujike, H., M. Takaki, M. Kodama, and S. Kuroda. 2002. Gene     expression related to synaptogenesis, neuritogenesis, and MAP kinase     in behavioral sensitization to psychostimulants. Ann N Y Acad Sci     965:55-67. -   10. Zhang, L., T. Schallert, Z. G. Zhang, Q. Jiang, P. Arniego, Q.     Li, M. Lu, and M. Chopp. 2002a. A test for detecting long-term     sensorimotor dysfunction in the mouse after focal cerebral ischemia.     J Neurosci Methods 117:207-14. -   11. Zhang, Z. G., L. Zhang, W. Tsang, H. Soltanian-Zadeh, D.     Morris, R. Zhang, A. Goussev, C. Powers, T. Yeich, and M. Chopp.     2002b. Correlation of VEGF and angiopoietin expression with     disruption of blood-brain barrier and angiogenesis after focal     cerebral ischemia. Cereb Blood Flow Metab 22:379-92.

Example 22 Telomerase Expression in umbilical-derived cells

Telomerase functions to synthesize telomere repeats that serve to protect the integrity of chromosomes and to prolong the replicative life span of cells (Liu, K, et al., PNAS, 1999; 96:5147-5152). Telomerase consists of two components, telomerase RNA template (hTER) and telomerase reverse transcriptase (hTERT). Regulation of telomerase is determined by transcription of hTERT but not hTER. Real-time polymerase chain reaction (PCR) for hTERT mRNA thus is an accepted method for determining telomerase activity of cells.

Cell Isolation. Real-time PCR experiments were performed to determine telomerase production of human umbilical cord tissue-derived cells. Human umbilical cord tissue-derived cells were prepared in accordance with the examples set forth above. Generally, umbilical cords obtained from National Disease Research Interchange (Philadelphia, Pa.) following a normal delivery were washed to remove blood and debris and mechanically dissociated. The tissue was then incubated with digestion enzymes including collagenase, dispase and hyaluronidase in culture medium at 37° C. Human umbilical cord tissue-derived cells were cultured according to the methods set forth in the examples above. Mesenchymal stem cells and normal dermal skin fibroblasts (cc-2509 lot # 9F0844) were obtained from Cambrex, Walkersville, Md. A pluripotent human testicular embryonal carcinoma (teratoma) cell line nTera-2 cells (NTERA-2 cl.Dl), (see, Plaia et al., Stem Cells, 2006; 24(3):531-546) was purchased from ATCC (Manassas, Va.) and was cultured according to the methods set forth above.

Total RNA Isolation. RNA was extracted from the cells using RNeasy® kit (Qiagen, Valencia, Calif.). RNA was eluted with 50 microliters DEPC-treated water and stored at −80° C. RNA was reverse transcribed using random hexamers with the TaqMan® reverse transcription reagents (Applied Biosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60 minutes and 95° C. for 10 minutes. Samples were stored at −20° C.

Real-time PCR. PCR was performed on cDNA samples using the Applied Biosystems Assays-On-Demand™ (also known as TaqMan® Gene Expression Assays) according to the manufacturer's specifications (Applied Biosystems). This commercial kit is widely used to assay for telomerase in human cells. Briefly, hTERT (human telomerase gene) (Hs00162669) and human GAPDH (an internal control) were mixed with cDNA and TaqMan® Universal PCR master mix using a 7000 sequence detection system with ABI prism 7000 SDS software (Applied Biosystems). Thermal cycle conditions were initially 50° C. for 2 minutes and 95° C. for 10 minutes followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute. PCR data was analyzed according to the manufacturer's specifications.

Human umbilical cord tissue-derived cells (ATCC Accession No. PTA-6067), fibroblasts, and mesenchymal stem cells were assayed for hTERT and 18S RNA. As shown in Table 22-1, hTERT, and hence telomerase, was not detected in human umbilical cord tissue-derived cells.

TABLE 22-1 hTERT 18S RNA Umbilical cells (022803) ND + Fibroblasts ND + ND—not detected; + signal detected

Human umbilical cord tissue-derived cells (isolate 022803, ATCC Accession No. PTA-6067) and nTera-2 cells were assayed and the results showed no expression of the telomerase in two lots of human umbilical cord tissue-derived cells while the teratoma cell line revealed high level of expression (Table 22-2).

TABLE 22-2 hTERT GAPDH Cell type Exp. 1 Exp. 2 Exp. 1 Exp. 2 hTERT norm nTera2 22.85 27.31 16.41 16.31 0.61 022803 — — 22.97 22.79 —

Therefore, it can be concluded that the human umbilical tissue-derived cells of the present invention do not express telomerase.

Example 23 hUTC in Rat Transient Middle Cerebral Artery Occlusion (MCAo) Stroke Model: Route of Administration Study

The study in this Example evaluated the efficacy of hUTC delivered by different routes of administration (intraarterial (IA), intracisterna magna (ICM), intrathecal (IT), and intracerebral (IC)) in a transient model of middle cerebral artery occlusion (tMCAo). Intravenous (IV) delivery was also examined as a positive control. Functional recovery was assessed at various time points over a 60-day period and compared to PBS controls. Histology, immunogenicity and tissue distribution of hUTC were also examined. Histology was performed on brain tissue to evaluate improve at the tissue and cellular levels. In addition, the production of antibodies against hUTC and the distribution of hUTC in tissues were evaluated.

Materials and Methods

Preparation of Test Article and Controls hUTC were isolated and cultured according to standard operating procedures (SOPs) and cryopreserved at ≦−120° C. in liquid nitrogen vapor phase. Cells were shipped to the test site in a liquid nitrogen dry shipper where they were thawed and processed on the day of administration.

The cells used in this study were of normal karyotype pathogen (HIV-1, HIV-2, CMV, HBV, HCV, HTL V, and EBV) free. Additionally, the cells were characterized for surface protein markers by flow cytometry as shown in Table 23-1.

TABLE 23-1 Surface protein markers for hUTC Fluores- Expres- Fluores- Expres- Antibody cence sion Antibody cence (%) sion IgGl-FITC 2.86 Neg CDl3-PE 263.53 Pos IgGl-PE 2.96 Neg CD10 62.37 Pos CD31-PE 1.71 Neg CD44-FITC 607.35 Pos CD34-FITC 2.31 Neg CD73-PE 221.66 Pos CD45R-PE 1.91 Neg HLA-ABC-PE 82.75 Pos CD117-PE 2.58 Neg PDGFr-a 21.44 Pos CD-141 1.98 Neg HLA-DPDQ 2.8 Neg Note: Neg = Negative; Pos = Positive

On the day of animal treatment, cells were thawed in a 37° C. water bath, washed with PBS (Invitrogen Corporation, Carlsbad, Calif.), and kept on ice prior to implantation. Viability was determined by trypan blue exclusion method. Cells with viability greater than or equal to 80% were utilized in this study.

Animals (Species, Weight, and Gender)

The Charles River Breeding Company (Portage, Mich.) supplied two to three month old male Wistar rats for use in this experiment. This species was selected to utilize an established method of inducing transient cerebral ischemic stroke. Approximately 10 to 15% more rats were ordered than needed for the stroke studies to permit replacement of animals that died following surgery. Rats were approximately 270 to 300 g at study initiation.

Animal Husbandry

The animals utilized in this study were handled and maintained according to protocols and guidelines approved by the Institutional Animal Care and Use Committee in accordance with the current requirements of the Animal Welfare Act (9 CFR) and the current standards set forth in the Guide for the Care and Use of Laboratory Animals.

Rats were housed singly in ventilated solid bottom cages and maintained on a 12-hr light/dark cycle with access to food (Harlan Teklad 22/5 Rodent Diet #8640) and municipal water ad libitum. Rats were handled daily to acclimate them to laboratory conditions and were tested in neurobehavioral tests prior to assignment to treatment. Each rat was given a unique identification number by tail marking. Extra rats were also handled as a contingency for potential animal mortality due to surgery.

Rats were observed for signs of pain/distress 2 times per day for 7 days, then 7 times per week for 4 weeks. Body temperature, vital signs, food intake and dehydration were monitored twice in the first 24 hours after surgery, another 2 times for the second 24 hours, and then once per day.

In the event of dehydration, an amount and duration of fluid (10 to 15 ml saline, bid/q.d.) was given based on the degree of dehydration. Apple slices or soft bacon was provided to rats to prevent severe bodyweight loss (more than 20%).

Allocation of Treatment Groups

Groups (10 treatment groups with 12 animals per group, 8 for behavior and histology; 4 for tissue distribution/antigenicity) of randomly selected animals with induced ischemic cerebral stroke were assigned to receive hUTC or vehicle by one of 5 routes of administration (see Table 23-2 below). Eight rats per group were assigned for behavioral efficacy and histology. To identify cell proliferation in the brain of these animals intraperitoneal injections of bromodeoxyuridine (BrdU, 50 mg/kg, Sigma, St. Louis Mo.) occurred daily for 14 consecutive days, starting 24 hrs after tMCAo.). Four rats per group were assigned to the antigenicity study in which the rats were assessed periodically for the development of antibodies to hUTC. At the termination of the study, organs from the antigenicity animals were used to examine tissue distribution of hUTC. Animals were sacrificed at day 60 post cell treatment. Both control vehicle and cell treatments were administered in an identical manner.

TABLE 23-2 Study Design Anti- Treat- Injec- Dose Functional genicity/ ment tion volume Recovery/ Tissue desig- Test Material Site (mL) Histo (n) dist (n) nation hUTC - 3 MM cells IV 2 8 4 Cell A hUTC - 2 MM cells IA 0.400 8 4 Cell A hUTC -1 MM cells ICM 0.040 8 4 Cell A hUTC -1 MM cells IT 0.040 8 4 Cell A hUTC -1 MM cells IC 0.012 8 4 Cell A PBS Ctrl IV 2 8 4 Cell B PBS Ctrl IA 0.400 8 4 Cell B PBS Ctrl IT 0.040 8 4 Cell B PBS Ctrl ICM 0.040 8 4 Cell B PBS Ctrl IC 0.012 8 4 Cell B IV = Intravenous; IA = Intraarterial; ICM = Intracisterna magna; IT = Intrathecal; IC = intracerebral (into the ipsilaterl hemisphere); Ctrl = control As used herein, the control was CalMg free PBS (Invitrogen, Carlsbad, CA Cat. #14190)

Test System

The model for cerebral stroke utilized male Wistar rats that had undergone temporary occlusion of the middle cerebral artery. Temporal middle cerebral artery occlusion (tMCAo) was induced in rats using a modified method of intraluminal vascular occlusion (Chen, H. et al. 1992. J. Cereb Blood Flow Meta 12:621-9). In brief, rats were anesthetized with 3.5% isoflurane in a jar for pre-anesthetic procedure followed by respiration of 1.5% isoflurane in 2:1 N₂O:O₂ mixture using a facemask connected and regulated with a modified FLUOTEC 3 Vaporizer (ASE, Atlanta, Ga.). The right common carotid artery, external carotid artery and internal carotid artery were exposed. A length of 3-0 monofilament nylon suture, varying between 18.5 to 19.5 mm depending on the animal weight, with its tip rounded by heating near a flame, was advanced from the external carotid artery into the lumen of the internal carotid artery until it blocked the origin of the MCA. Two hours after MCAo, reperfusion was permitted by withdrawal of the suture until the tip cleared the internal carotid artery thereby creating the tMCAo. The described treatments were administered on the day following tMCAo.

Experimental Design

Ischemic cerebral stroke was induced in 8 male Wistar rats per day, which were then placed on study equally across all treatment groups. On the day following tMCAo, rats were administered either PBS or hUTC cells by the routes of administration described below. Cell dose determination was estimated based on the principal investigator's experience testing mesenchymal stem cells in this model and from similar work published in the field (Seyfried, D M et al. 2008. Brain Res. 1224:12-9; Zhang, Z G. et al. 2003. Ann Neurol. 53(2): 259-63; Li, Y. et al. 2000. J Cereb Blood Flow Metab. 9:1311-9; Chen, J. et al. 2000. Neuropharmacology. 39(5):711-6; Lepore, A C et al. 2005. Brain Res. 1045:206-16; Chen, J. et al. 2001. Stroke 32: 1005-11).

IV Injections

At 1 day after tMCAo, animals were anesthetized with 3.5% isoflurane in a jar for pre-anesthetic procedure followed by respiration of 1.5% isoflurane in 2:1 N₂O:O₂ mixture using a facemask connected and regulated with a modified FLUOTEC® 3 Vaporizer (ASE, Atlanta, Ga.). A 27-gauge needle was inserted into the tail vein at 3-6 cm from the base of the tail. Accurate placement is confirmed by an aspirate of blood into the syringe. Approximately 3×10⁶ hUTC in a total volume of 2 ml were injected into the tail vein.

IA Injections

At 1 day post-ischemic insult, selected tMCAo rats were administered cells via IA route of administration. Briefly, the rat was anesthetized and a modified polyethylene catheter (PE-50, Becton Dickinson, Sparks, Md.) with a 0.3 mm outer diameter was advanced through the same small puncture in the external carotid artery (ECA) used for the embolizing nylon suture. It was passed into the lumen of the internal carotid artery (1CA) for a distance of ˜15.0 mm, lodging ˜2 mm proximal to the origin of the tMCA. The external end of the catheter port was connected to a syringe with a 23-gauge needle. Approximately 2×10⁶ hUTC in 400 μl PBS or vehicle control (400 μl PBS) was slowly injected over a 5-minute period into each rat. The catheter was withdrawn immediately after injection, and the right ECA was ligated.

ICM Injections

At 1 day post-ischemia, anesthetized rats were placed on ear bars of a stereotaxic frame (Model 51603, Stoelting Co., Wood Dale, Ill.). The back of the head and neck were shaved and draped to create a sterile field. The head was positioned straight down in the earbars (in the vertical plane). This created a 90° angle with respect to the body, which remained in the horizontal plane. The space between the base of the skull and the spine marked the site of entry. A 100 μl Hamilton (Reno, Nev.) syringe with a 22-gauge needle was advanced through the skin at the level of the cisterna magna, until the dura was punctured (usually between 7.0 and 9.5 mm deep from skin level). Correct positioning of the needle into the cisterna magna was determined by the reflux of cerebrospinal fluid (˜2 μl CSF) into the syringe. 40 μl of PBS containing approximately 1×10⁶ cells were injected, and the needle withdrawn immediately after injection.

IT Injections

At 1 day post-ischemia, selected tMCAo rats were anesthetized and placed on an operating surface that flexes the animal's back. A small (1 cm) longitudinal incision was made over the L4-5 spinous processes, and the skin was retracted. At the L4/L5 intervertebral space, the dura was exposed. A Hamilton syringe equipped with a 22-gauge needle was inserted between the L4/L5 vertebrae above the cauda equina. Intrathecal placement was verified by a flick of the tail on needle entry. 40 μl of PBS containing approximately 1×10⁶ cells were injected, and muscle and skin was sutured immediately after injection.

IC Injections

Anesthetized rats were mounted on a stereotaxic apparatus (Model 51603, Stoelting Co., Wood Dale, Ill.) 1 day post-ischemic insult. Using aseptic technique, a 26-gauge Hamilton syringe containing cells (1×10⁶) in a volume of 12 μl was inserted vertically through a burr hole (1 mm) into the skull and into the right striatum at the coordinates 2.0 mm lateral to the midline (LM), 4.5 mm in the vertical depth (VD) and anterior-posterior (AP) relative to the bregma, and into the right cortex at LM=2.0 mm, VD=2.0 mm and AP=0 mm (Paxinos G. and C. Watson. 1986. The rat brain in stereotaxic coordinates, 2^(nd) ed. Sydney: Orlando: Academic Press). This position is in the approximate region of the ischemic boundary zone (IBZ). First, the needle was advanced to the position in the striatum where 8 μA of cells was injected. In the same vertical track the needle was retracted back to the position of the cortex where the remaining 4 μl was injected. This was conducted over a period of 10 minutes in each region. The needle was retained in the cortex for an additional 5-minute interval to avoid cell reflux from the injected areas to the brain surface. After injection, bone wax (W810; Ethicon, Somerville, N.J.) was placed on the skull to prevent leakage of the solution.

Neurological Functional Tests

Three behavioral tests were performed 1-day after tMCAo, but prior to treatment and weekly (Days 7, 14, 21, 28, 35, 42, 49, 56 and 60) thereafter by an investigator blinded to the experimental groups.

Modified neurological severity score (mNSS) is a composite of motor, sensory, balance and reflex tests (Chen, J. et al. 2001 Stroke 32:1005-11). Neurological function was graded on a scale of 0 to 18 (normal score 0; maximal deficit score 18) with one point awarded for the exhibition of specific abnormal behavior or for lack of a tested reflex. A greater impairment of normal function resulted in a higher score.

In the foot-fault test, rats were tested for placement dysfunctions of forelimb (Zhang, L. et al. 2002a J. Neurosci Methods 117:207-14). Rats were placed on elevated hexagonal grids of different sizes. The number of steps used to cross the grid was counted up to 100 steps or ten minutes, whichever came first, and the total number of foot faults for each forelimb was recorded. Foot-fault values are presented as the number of missteps of the contralateral forelimb divided by the total number of steps counted. It is well established that in animals subjected to tMCAo, the impaired (contralateral) limbs fault more often than non-impaired limbs.

The adhesive patch removal test requires the use of adhesive-backed paper dots of equal size (113.1 mm²) as bilateral tactile stimuli on the distal-radial region of each forelimb (Schallert, T. et al. 1997 Adv Neurol. 73:229-38). Prior to surgery rats were trained for 3 days to remove the patches. Once the rats were able to remove the adhesive dots within 10 seconds, they were subjected to tMCAo. Post-tMCAo, the time to remove each stimulus from both forelimbs was recorded on three (3) trials per day. Individual trials were separated by at least 5 minutes.

Brain Section Preparation and Evaluation

At study termination, rats were administered Ketamine (160 mg/kg) and Xylazine (26 mg/kg) via intraperitoneal injection. Rats were exsanguinated under deep anesthesia by piercing the right atrium as the brains were fixed by transcardial (left ventricle) perfusion with saline, followed by perfusion and subsequent immersion in 4% paraformaldehyde. Using a rat brain matrix (Activational Systems Inc., Warren, Mich.), each forebrain was cut into 2-mm thick coronal blocks for a total of 7 blocks from bregma 5.2 mm to bregma −8.8 mm per animal (Paxinos G. 1986). The tissue blocks were embedded in paraffin, and a series of 6 μm-thick sections were cut corresponding to coronal coordinates and placed on microscope slides. One of each of the coronal paraffin slides (6 μm thick) collected from the seven blocks was stained with hematoxylin and eosin (H&E). These slides were traced using the Global Lab Image analysis system (Data Translation, Marlboro, Mass.).

Light and fluorescent microscopy was performed using an Olympus BH-2 microscope (Olympus, Olympus America Inc., 3500 Corporate Parkway, P.O. Box 610, Center Valley, Pa. 18034-0610).

Histological Assessments

Reagents used for analyzing brain cell structures and characteristics are listed in Table 23-3 shown below:

TABLE 23-3 Histochemical Reagents Reagent Catalog # Vendor BrdU B5002 Sigma Mouse anti-BrdU M0744 Dako Rabbit anti vWF AOO82 Dako Mouse anti synaptophysin MAB5258 Chernicon FITC conjugated antimouse IgG 66581 Jackson Immunoresearch FITC conjugated anti rabbit IgG 59461 Jackson Immunoresearch CY3 conjugated anti mouse IgG 63394 Jackson Immunoresearch ABC kit PK-6100 Vector Labs 3,3- Diarninobenzidine D5637 Sigma tetrahydrochloride hydrate (DAB) in situ Apoptosis Detection kit S7100 Chernicon

Measurement of Infarct Area

Lesion area was measured indirectly by subtracting the intact area of the ipsilateral hemisphere from the area of the contralateral hemisphere (Swanson, R. et al. 1990. J Cereb Blood Flow Metab 10:290-3). Lesion volume is presented as a volume percentage of the lesion compared to the contralateral hemisphere.

Immunohistochemical Assessment

Paraffin sections corresponding to coronal coordinates for bregma −1 to ˜1 mm were used for this analysis. To distinguish proliferating cells, a mouse antibody against BrdU (Dako Glostrup, Denmark) was applied at a titer of 1: 100 (Chen J. 2001. Stroke 32:1005-11). Moreover, antibodies against von Willebrand factor (vWF, 1:400, Dako) (Chen, J. et al. 2004. Brain Res. 105:21-8) and synaptophysin (1:500, Chemiconi) (Chen, J. et al. 2003. Ann. Neurol. 53:743-51) were used to identify vessels and synapses, respectively.

Apoptotic Cell Staining

The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method (in situ Apoptosis Detection Kit, Chemicon 28820 Single Oak Drive, Temecula Calif. 92590) was used to identify and assess apoptotic cells in situ (Li, Y. et al. 1998. J Neurol Sci 156:119-32; Li, Y. et al. 1995. Am J Pathol 146:1045-1051). The TUNEL method is based on the specific binding of TdT to 3′-OH ends of DNA and the ensuing synthesis of polydeoxynucleotide polymer cells. Staining was performed according to procedures provided by the manufacturer.

Cell Quantification

BrdU-positive, proliferating, cells were identified by their brown color and presented as number of cells per mm² in the subventricular zone (SVZ). The number of BrdU positive cells in both ipsilateral and contralateral SVZ wall was counted. Data were presented as the average number of BrdU positive cells per mm².

For measurement of vascular density, apoptotic cell number and synapse density, 8 fields of view from the ischemic boundary zone (IBZ, FIG. 5) were digitized using a x40 objective via the MicroComputer Imaging Device (MCID) system. The numbers of vWF imunnoreactive vessels, TUNEL positive cells, or the positive area of synaptophysin in the IBZ were measured throughout each field of view. Data were presented as density of vWF imunnoreactive vessels (vessels/mm²), density of TUNEL positive cells (cells mm²), and percentage of synaptophysin immunoreactive area, respectively. For measurement of vascular perimeter, 10 enlarged vessels in the ipsilateral brain were digitized under a 40× objective. Data were presented as total perimeter of 10 enlarged vessels (μm). The number of BrdU positive cells in ipsilateral SVZ were digitized in the same manner described above.

Tissue Distribution

Sample Preparation

Animals were sacrificed at day 60 by deep dose of anesthesia (Ketamine (160 mg/kg) and Xylazine (26 mg/kg)) (IP) followed by exsanguination. The brain (bilateral hemispheres), kidney (bilateral), heart, liver, spleen and lung were harvested from each rat. The organs were snap frozen and stored at −80° C. until DNA was isolated.

DNA Extraction/Sample Preparation

On the day of DNA extraction, the frozen whole organs were thawed in tubes on wet ice and homogenized with a chilled mortar and pestle. Following complete homogenization of the organ, a 25 mg sample was processed for DNA extraction employing a DNeasy® Tissue Kit (Qiagen, catalog #69506) according to the manufacturer's protocol (except for the spleen where a 10 mg sample was processed). Briefly, the homogenate was lysed by Proteinase K digestion for 3 hours at 56° C. Following lysis, RNase A was added to the sample to prevent the isolation of RNA with the genomic DNA. The genomic DNA was purified via a spin-column method and eluted from the column with 100 μl of elution buffer. A spectrophotometer was employed to determine the DNA concentration and samples were stored at −20° C. until further use.

Probe Generation/Labeling

Primers and probes for the ERV TaqMan PCR were designed III collaboration with CBAT, and were:

ERV-3 F: CATGGGAAGCAAGGGAACTAATG (SEQ ID NO: 11) ERV-3 R: CCCAGCGAGCAATACAGAATTT (SEQ ID NO: 12) ERV-3 Probe: TCTTCCCTCGAACCTGCACCATCAAGTCA (SEQ ID NO: 13) The probe was custom labeled with RAM (Ramification amplification) and TAMRA (N,N,N′, N′-tetramethyl-6-carboxyrhodamine) fluorochromes (manufactured by Applied Biosystems).

PCR Parameters

An ABI Prism 7000 Sequence Detection machine was used for real time PCR experiments (Instrument Serial #: 270002086). All samples were run in triplicate in a MicroAmp® Optical 96-Well Reaction plate (Applied Biosystems Cat #: N8010560). The PCR reaction comprised: 0.2 μl probe; 1 μl for each primer; 10 μl TaqMan master mix (Applied Biosystems cat#: 4364338); and 8 μl of DNA (1 μg). PCR was performed using the following cycling conditions: (a) 1 cycle at 95° C. for 10 minutes; (b) 40 cycles at 95° C. for 15 seconds and (c) 60° C. for 1 minute.

DNA Optimal Concentrations

DNA isolated from hUTC was used to determine the optimal DNA concentration for PCR. DNA was serially diluted from a 2.0 μg stock and real time PCR was performed as previously described. Based on these data, an optimal working concentration of DNA was determined. All test samples were prepared based on this concentration (1 μg).

Standard Curve:

hUTC were serially diluted (1:2) from 40,000 cells to ˜3 cells in autoclaved ddH₂0 to generate fifteen standard curve samples. Water was used as a negative control. Genomic DNA was isolated from all fifteen samples using a DNeasy® Tissue Kit and the PCR reaction was run as previously described. The Ct values from the standard curve samples were obtained and an inverse power function was used to create a trendline. All predicted cell numbers from test samples were calculated using this equation and presented as cell number/whole organ.

Agarose Gel Confirmation

All PCR results were confirmed by running the TaqMan PCR products in 2% agarose gels with ethidium bromide. The total volume of each sample well was loaded into the agarose gels and visualized under UV light. Only those samples that had both a positive Ct value and a visible band in the agarose gel were accepted as a positive result.

Serum Antigenicity Analysis

Blood was drawn from cohorts at Day 7, Day 14, Day 28, and Day 60 post cell injection. At least 0.5 mL of blood was taken from the tail vein using an IV catheter needle (BD Angiocath 24GA 0.751N Ref # 381112) and collected in a plasma separator tube (BD Microtainer, Catalog #365965). The tubes were centrifuged and the serum collected into labeled storage vials (snap tubes or cryovials) so each animal may be tracked. The samples were stored frozen at approximately −70° C. The serum was then assessed for the production of rat antibodies to hUTC.

Serum Antibody FACS

Human hUTC 120304 were seeded from thaw at 5,000 per cm² on T-75 tissue culture flasks (Costar, Cat #430641) in Hayflick growth media. Cells were incubated at 37° C., 5.0% CO₂ in a humidified incubator. After 3 days, recombinant human IFN-gamma was added to the hUTC at 500 U/ml (25 ng/ml) to induce MHC class II expression. The hUTC were cultured for an additional 48 hours. All the cells were then washed with PBS and detached from the flask using TrypLE™ Select. Detached cells were washed with media and centrifuged at 250×g for 5 minutes and resuspended in sheath fluid (Fluorescence Activated Cell Sorting (FACS) buffer) containing 3% Fetal Bovine Serum (FBS). Serum samples drawn from the experimental animals were heat-inactivated for 30 minutes at 56° C. 10 μl of each serum sample was mixed with activated and unactivated hUTC in separate FACS tubes, incubated for 30 minutes at 4° C. and then washed with sheath fluid and 3% fetal bovine serum. 2 μl of anti-rat IgG1+2a fluorescein isothiocyanate (FITC; BD Pharmingen, 553881) and 2 μl of anti-rat IgM PE (BD Pharmingen, 553888) secondary antibodies were added to each tube and incubated for 30 minutes at 4° C. and then washed with sheath fluid containing 3% FBS. Cells were brought to a final volume of 500 μl sheath fluid and fluorescence values were acquired using a flow cytometer.

Data Analysis

Comparisons of histology measures were made between treatment groups by administration route using two-sample Student's t-tests. Analysis of variance for repeated measures with fixed factor treatment hUTC and repeated factor time was used to compare behavioral measures between groups. Pair-wise comparisons were made between treatment groups by time if there was a significant interaction between hUTC and time (p<0.10) or a significant main effect for group (p<0.05). Body weight at baseline was compared between treated and control animals within each route of administration group using two-sample t-tests. Repeated measures analysis of covariance (ANCOVA) was used to test for differences in body weight by time between treated and control animals within each ROA group, adjusting for baseline weight. Behavioral data were evaluated for normality and the nonparametric analysis approach was considered if data did not follow a normal distribution. Histology data was evaluated using Student's t-test. Differences were considered significant when the probability of making a type 2 error was less than or equal to 5%, p≦0.05. Differences in body weight between treatment groups were analyzed by Student's t-test at each collection interval. Data are presented as mean values±SD (standard deviation).

Results

Mortality

130 animals were employed for transient middle cerebral artery occlusion (tMCAO) in this experiment of which 10 rats died. Eight rats died within 1 day after tMCAo but before the initiation of treatment. One rat, which received vehicle (in the IV cohort), was found dead four days after tMCAo due to subarachnoid hemorrhage. One rat, which received hUTC (in the ICM cohort) expired ten minutes after cell injection due to brain stem damage. It is noted that the reported mortality rate for tMCAo in Wistar rats varies greatly among different laboratories (Kollmar R. et al. 2002. Stroke 33:1899-1904; Matsou, Y. et al. 2001. Stroke 32:2143-48; Mokudai, T. et al. 2000. Stroke 31: 1679-85; Pfefferkorn, T and Rosenberg G A. 2003. Stroke 34:3025-2030). In the laboratory used for this study, the mortality rate after tMCAo ranges from 5 to 20%.

Body Weight

A summary of body weights is presented in Table 23-4 with significant differences between hUTC and control treatment (p<0.05) indicated by shaded “p-values”. hUTC treated rats were significantly heavier than control animals at times after intraarterial (Days 28-49), intracerebral (Days 28-60) or intravenous (Days 14-60) administration. Body weights did not differ between hUTC and control treated groups at any time interval following ICM and IT administration.

TABLE 23-4 Results for Body Weight by Group, Treatment Mode, and Time Cell A (hUTC) Cell B (PBS) Mode Time Mean SD Mean SD p-value* IA Base 284.38  6.61 286.63  9.10 0.581   1 252.88  6.73 255.25 13.38 0.952   7 251.50 13.88 241.75 13.10 0.308  14 272.25 18.80 255.00 17.85 0.112  21 300.63 22.75 279.38 28.49 0.060  28 344.13 15.12 309.50 35.26

35 366.88 21.92 334.00 39.95

42 383.25 23.86 354.75 42.76

49 398.88 20.75 376.50 40.70

56 411.50 19.05 391.38 41.57 0.072  60 420.63 18.75 402.25 45.06 0.095  IC Base 289.25 11.02 293.38 12.03 0.486   1 255.75 13.71 264.13 17.12 0.600   7 248.88 22.52 250.25 20.24 0.798  14 271.00 23.95 264.63 18.88 0.267  21 293.25 25.60 283.00 21.23 0.128  28 320.88 25.57 302.75 20.08

35 344.25 24.36 320.63 21.80

42 365.75 21.05 337.25 19.14

49 384.50 21.79 354.88 20.41

56 405.25 21.35 371.38 19.87

60 412.63 22.93 383.38 18.21

ICM Base 290.13  9.75 287.13 8.13 0.515   1 256.00 16.41 257.25 11.42 0.565   7 254.25 16.15 246.88 15.90 0.884  14 269.25 15.55 255.63 16.85 0.505  21 298.63 18.16 278.75 17.30 0.239  28 326.88 20.48 302.50 23.85 0.125  35 348.00 14.03 331.00 35.67 0.345  42 364.00 11.26 346.63 35.29 0.330  49 375.88 14.24 362.00 38.14 0.492  56 391.88 24.97 378.38 43.17 0.512  60 409.25 37.19 391.88 41.71 0.330  IT Base 282.25  5.90 278.75 10.10 0.411   1 242.38  7.33 244.13 10.33 0.917   7 238.75 25.87 240.75 25.00 0.904  14 266.50 36.22 262.38 33.85 0.787  21 289.88 34.59 279.38 34.46 0.500  28 315.13 35.61 302.00 33.77 0.401  35 337.25 36.88 323.13 38.56 0.367  42 359.25 34.09 343.50 41.29 0.316  49 380.75 35.05 360.50 40.16 0.201  56 398.00 33.39 379.13 40.17 0.232  60 405.50 33.17 387.88 38.39 0.263  IV Base 286.50  8.70 287.75  8.29 0.773   1 256.88  9.55 257.00 13.82 0.872   7 249.63 11.19 236.50 18.65 0.184  14 273.00 21.73 249.00 17.63

21 297.75 29.94 268.88 25.72

28 328.38 31.12 294.25 21.87

35 354.75 33.76 322.00 24.10

42 375.13 40.18 349.88 18.67

49 390.63 39.19 362.13 23.76

56 410.13 38.66 380.00 25.54

60 420.00 38.19 389.75 22.14

*Baseline P-value calculated from two-sample t-test. Statistically significant values are highlighted in gray P-values for rest of time intervals calculated from repeated measures ANCOVA model adjusted for baseline bodyweight

Behavioral Testing

Tables 23-6 to 23-8 present the mean, standard deviation and p-value from the pair-wise comparisons of treatment group for mNSS, adhesive test and foot-fault respectively for Days 1, 7, 14, 21, 28, 35, 42, 49, 56 and 60. Significant (p<0.05) improvement in behavioral test scores is shown in Table 23-5 by shaded entries. There was no significant improvement in test scores earlier than Day 7 regardless of the route of administration. The intravenous route of administration resulted in the most consistent recovery response attributable to the hUTC across all three behavioral tests. The intracerebral route was the second-most consistent (Table 23-5 for data review and Tables 23-6 to 23-8 for complete data sets).

TABLE 23-5 Significant differences in behavioral parameters between hUTC- and control-treated rats from selected test days for three behavioral tests across the various routes of administration Behavior Day of Route of Administration Parameter Test IA IC ICM IT IV mNSS 14

NS

28

56 NS

NS

60 NS

NS NS

Adhesive 14

NS *

28

NS *

56 NS

NS NS

60 NS

NS NS

Foot-Fault 14 NS NS NS

28

NS

56

NS

60

NS = not statistically significant.

Modified Neurological Severity Score Test (mNSS) Test

The mNSS test is an 18-point scale that measures motor, reflex and balance deficits over the course of recovery. Higher scores reflect greater functional impairment. The results for the mNSS test are shown in Table 23-6 below.

TABLE 23-6 Results for mNSS by Group, Treatment Mode, and Time Cell A (hUTC) Cell B (PBS) Mode Time Mean SD Mean SD p-value* IA 1 10.75 0.89 10.63 1.19 0.825 7 9.88 1.13 10.13 1.13 0.659 14 8.50 0.76 9.75 1.28 0.032 21 7.50 0.76 9.00 1.20 0.011 28 6.88 0.99 8.38 1.19 0.011 35 6.50 0.93 7.88 1.25 0.019 42 6.00 1.07 7.38 1.41 0.019 49 5.63 0.74 6.88 1.13 0.032 56 4.75 1.04 5.50 1.41 0.191 60 4.75 1.04 5.50 1.41 0.191 IC 1 10.88 0.64 1138 0.92 0.211 7 10.50 0.76 10.50 0.76 0.999 14 8.88 0.99 9.63 0.74 0.063 21 7.13 0.64 8.88 0.99 <.0001 28 6.38 1.06 8.13 0.99 <.0001 35 6.00 0.76 7.38 0.74 0.0009 42 5.50 0.53 6.75 0.89 0.0025 49 5.25 0.46 6.13 0.83 0.031 56 4.75 0.71 5.75 0.71 0.014 60 4.38 0.74 5.38 0.74 0.014 ICM 1 10.88 0.35 10.75 0.71 0.767 7 9.88 0.83 10.00 0.76 0.767 14 8.63 0.74 9.63 0.74 0.021 21 7.75 0.89 8.88 0.64 0.010 28 7.25 0.71 8.38 0.52 0.010 35 5.88 0.99 8.00 0.76 <.0001 42 5.38 0.92 7.25 139 <.0001 49 5.25 1.04 6.50 0.93 0.004 56 4.88 0.83 5.88 1.13 0.021 60 4.75 0.89 5.50 0.93 0.080 IT 1 10.75 0.89 10.88 0.64 0.784 7 10.38 0.74 10.38 0.74 0.999 14 8.75 139 9.75 1.16 0.032 21 7.50 1.20 9.00 1.20 0.0018 28 6.75 0.89 8.25 1.04 0.0018 35 6.00 1.07 7.75 1.28 0.0003 42 5.75 0.89 7.38 1.19 0.0008 49 5.50 0.76 6.63 0.74 0.0167 56 5.25 0.46 5.88 0.64 0.175 60 5.00 0.76 5.75 0.71 0.105 IV 1 10.75 0.71 10.88 0.83 0.824 7 9.38 1.19 10.25 1.04 0.124 14 8.25 1.16 9.63 130 0.018 21 7.38 130 9.13 1.55 0.003 28 6.50 1.07 8.63 1.51 0.0005 35 6.13 1.25 7.88 1.46 0.003 42 5.50 131 7.50 131 0.0009 49 4.75 1.04 6.63 1.51 0.0017 56 4.50 0.93 6.13 1.25 0.0057 60 4.38 0.92 5.63 1.06 0.030 *P-value calculated from pair-wise comparisons from repeated measures ANOVA models..

For the IA route, significant differences were observed in rats receiving hUTC at day 14, 21, 28, 35, 42, and 49 following tMCAo, as compared to the PBS control (see FIG. 6A). For the IV route, significant differences were observed between rats receiving hUTC and vehicle starting at day 14, which persisted up to 60 days post-tMCAo (see FIG. 6B). For the ICM route, significant differences were observed between rats receiving hUTC at day 14, 21, 28, 35, 42, 49, and 56 following tMCAo, as compared to the PBS control (see FIG. 7A). For the IT route, significant differences were observed between rats receiving hUTC at day 14, 21, 28, 25, 42 and 49 following tMCAo, as composted to PBS control (see FIG. 7 B). For the IC route, significant differences were observed between rats receiving hUTC and vehicle starting at day 21 which persisted up to day 60 after tMCAo (see FIG. 8).

Adhesive Patch Removal Test

The adhesive patch removal test measures the amount of time it takes for the animals to remove an adhesive patch from its forepaws. The results for the adhesive removal patch test are shown in Table 23-7.

TABLE 23-7 Results for Adhesive Removal Patch Test by Group, Treatment Mode, and Time Cell A (hUTC) Cell B (PBS) Mode Time Mean SD Mean SD p-value* IA 1 117.67 4.55 115.79 7.00 0.740 7 114.13 11.66 116.00 5.34 0.740 14 9508 12.40 112.75 8.87 0.002 21 8608 10.97 97.54 12.37 0.0045 28 72.13 9.19 90.25 12.78 0.0018 35 61.38 11.36 77.96 12.17 0.004 42 49.88 7.57 68.38 8.30 0.0015 49 40.33 5.40 36.46 24.25 0.493 56 29.54 7.29 33.42 18.04 0.493 60 21.46 4.45 27.33 14.58 0.299 IC 1 116.67 5.46 117.63 3.51 0.761 7 111.21 5.29 111.83 6.24 0.843 14 95.75 5.95 104.79 4.91 0.005 21 81.63 706 90.75 4.22 0.005 28 71.04 7.49 8308 6.87 0.0003 35 50.83 8.65 70.25 8.36 <.0001 42 44.50 6.78 63.42 10.41 <.0001 49 35.92 5.05 49.54 8.18 <.0001 56 25.54 4.24 3508 5.95 0.003 60 20.88 3.87 28.13 5.76 0.024 ICM 1 115.17 5.88 117.13 5.49 0.690 7 110.88 9.11 111.17 10.89 0.953 14 98.58 9.90 104.00 9.97 0271 21 82.33 608 92.63 11.94 0.039 28 75.42 12.82 8308 1202 0.121 35 52.25 6.68 71.21 11.84 0.0002 42 4304 770 56.21 10.64 0.009 49 33.25 4.67 41.00 10.65 0.117 56 25.63 6.12 30.88 16.62 0.286 60 18.75 8.84 2604 11.90 0.140 IT 1 113.21 5.20 114.75 5.33 0.647 7 107.79 4.34 111.67 6.24 0.253 14 93.92 6.67 105.29 5.49 0.001 21 81.08 11.62 94.29 6.51 0.0002 28 70.29 9.31 85.00 6.88 <.0001 35 55.04 8.91 76.42 5.85 <.0001 42 48.38 9.35 6504 4.58 <.0001 49 36.50 770 45.88 3.24 0.007 56 25.92 7.35 32.46 5.02 0.056 60 21.04 7.52 25.17 4.65 0.224 IV 1 114.29 8.50 116.79 9.07 0.584 7 106.13 8.49 115.42 9.94 0.044 14 89.67 12.05 110.71 11.99 <.0001 21 71.96 8.10 99.75 9.28 <.0001 28 55.63 6.69 92.33 9.87 <.0001 35 47.21 5.46 79.13 9.10 <.0001 42 37.83 5.62 60.00 12.82 <.0001 49 27.79 7.26 44.63 12.34 0.0004 56 2404 8.47 37.75 10.35 0.0035 60 19.33 6.52 3008 7.24 0.021 *P-value calculated from pair-wise comparisons from repeated measures ANOVA models.

For the IA route, significant differences were observed between rats receiving hUTC at 14, 21, 28, 35, and 42 days following tMCAo, as compared to the PBS control (see FIG. 9A). For the IV route, significant differences were observed between rats receiving hUTC and vehicle starting at day 7 which persisted up to day 60 after tMCAo (see FIG. 9B). For the ICM route, significant differences were observed between rats receiving hUTC and vehicle at day 21, 35, and 42 after tMCAo (see FIG. 10A). For the IT route, significant differences were observed between rats receiving hUTC at day 14, 21, 28, 35, 42, and 49 following tMCAo, as compared to the PBS control (see FIG. 10B). For the IC route, significant differences were observed between rats receiving cell A (hUTC) and B (vehicle) starting at day 14 which persisted up to day 60 after tMCAo (see FIG. 11).

Foot-fault test

In the foot-fault test, rats were tested for placement dysfunctions of the forelimb. The results for the foot-fault test are shown in Table 23-8 below:

TABLE 23-8 Results for Foot-fault by Group, Treatment Mode, and Time Cell A (hUTC) Cell B (PBS) Mode Time Mean SD Mean SD p-value* IA 1 35.76 4.72 36.03 3.74 0.859 7 29.13 2.59 30.75 406 0.290 14 26.00 1.51 28.50 3.34 0.107 21 22.13 2.10 26.00 3.42 0.015 28 19.00 2.39 22.75 2.82 0.018 35 16.38 3.34 21.13 2.75 0.0034 42 15.00 2.73 20.00 2.73 0.0022 49 13.50 2.51 17.75 2.43 0.008 56 11.50 2.27 16.88 2.47 0.0011 60 10.75 1.98 15.75 1.98 0.0022 IC 1 35.91 3.63 36.49 3.84 0.753 7 30.00 5.55 31.00 4.14 0.587 14 25.13 3.18 27.25 3.54 0.253 21 20.88 402 24.75 403 0.042 28 17.88 3.40 22.25 3.85 0.023 35 15.75 3.85 20.25 406 0.020 42 14.00 3.55 18.38 3.81 0.023 49 12.25 3.37 17.13 3.76 0.012 56 11.00 302 15.38 3.58 0.023 60 10.13 2.80 13.88 3.60 0.049 ICM 1 35.76 3.63 37.33 4.31 0.416 7 29.63 3.46 30.25 4.27 0.745 14 24.50 2.88 27.25 3.41 0.160 21 21.50 2.98 25.00 3.46 0.077 28 18.63 2.50 22.50 3.51 0.052 35 16.88 2.75 20.00 3.12 0.112 42 15.13 2.36 18.63 3.78 0.077 49 13.88 2.53 17.00 4.75 0.112 56 12.13 2.70 15.13 601 0.127 60 9.63 2.56 14.25 5.04 0.022 IT 1 36.38 302 36.75 2.66 0.790 7 29.38 3.66 30.88 3.64 0.291 14 25.63 3.25 27.88 3.18 0.117 21 21.00 2.45 25.25 2.31 0.005 28 18.25 2.31 22.00 2.83 0.012 35 16.25 2.05 20.00 3.12 0.012 42 14.88 2.10 18.13 2.95 0.027 49 13.00 185 16.88 3.23 0.009 56 1188 2.03 15.50 3.16 0.014 60 10.38 1.69 14.25 3.41 0.009 IV 1 34.53 4.72 36.75 4.68 0.197 7 30.13 3.80 31.50 3.63 0.419 14 24.88 2.47 28.50 3.16 0.042 21 21.00 3.21 26.00 2.67 0.007 28 17.38 2.83 22.88 2.42 0.0035 35 15.25 2.12 21.13 2.64 0.0021 42 14.25 1.49 19.25 2.60 0.007 49 12.25 1.49 17.75 2.49 0.0035 56 11.50 1.60 17.00 2.20 0.0035 60 10.50 1.93 15.50 2.33 0.007 *P-value calculated from pair-wise comparisons from repeated measures ANOVA models.

For the IA route, significant differences were observed between rats receiving cell A (hUTC) and B (vehicle) starting at day 21 which persisted up to day 60 after tMCAo (see FIG. 12A). For the IV route, significant differences were observed between rats receiving hUTC and vehicle starting at day 14 which persisted up to day 60 after tMCAo (see FIG. 12B). For the ICM route, significant differences were observed between rats receiving hUTC and vehicle at 60 days-post tMCAo only (see FIG. 13A). For the IT route, significant differences were observed between rats receiving hUTC and vehicle starting at day 21 which persisted for up to day 60 after tMCAo (see FIG. 14B). For the IC route, significant differences were observed between rats receiving hUTC and vehicle starting at day 21 which persisted up to day 60 after tMCAo (see FIG. 15).

Histological Findings

Tables 23-9 to 23-14 present the mean, standard deviation, and p-value from t-test comparisons of histological findings for rats by route of administration. Significant (p<0.05) improvement in histological parameters is shown in Table 23-9 by shaded entries.

TABLE 23-9 Summary of significant differences in histological parameters between hUTC- treated and control-treated rats across route of administration cohorts Histological Route of administration parameter IA IC ICM IT IV BrdU Contralateral^(a) NS NS NS NS NS BrdU Ipsilateral^(b)

Lesion volume^(c) NS NS NS NS NS Synaptophysin^(d)

NS

TUNEL (apoptosis)^(e)

NS NS

vWF Perimeter^(f) NS

NS NS

vWF vessel^(g)

Complete Data in: Table 23-10 Table 23-11 Table 23-12 Table 23-13 Table 23-14 ^(a)Extent of BrdU staining on the ipsilateral SVZ in cells per mm² ^(b)Extent of BrdU staining on the contralateral SVZ in cells per mm² ^(c)Lesion volume is expressed in percent compared to the contralateral section of the brain. ^(d)Synaptophysin, a marker for synapses, is expressed in % in IBZ ^(e)Apoptosis is expressed as the number of TUNEL+ cells per mm² ^(f)Total perimeter of 10 vWF positive vessels in a 40× microscopic field (μm) ^(g)Density of vWF vessels in IBZ (vessels/mm²) *indicates significant at p < 0.05

TABLE 23-10 Student's t-test comparisons of histology measures - IA route Cell A (hUTC) Cell B (PBS) Variable (N = 8) (N = 8) p-value BrdU Contralateral^(a) 213.9 ± 82.8 172.4 ± 82.0 0.331 BrdU Ipsilateral^(b)  605.9 ± 134.7  349.9 ± 161.1 0.004* Lesion volume^(c)  31.5 ± 11.9  31.5 ± 10.7 1.000 Synaptophysin^(d) 16.8 ± 4.9 12.5 ± 2.3 0.045* TUNEL (apoptosis)^(e)  21.9 ± 11.0  45.0 ± 27.3 0.043* vWF Perimeter^(f) 2417.6 ± 792.9 1840.7 ± 470.0 0.098 vWF vessel^(g) 262.0 ± 43.0 223.6 ± 25.4 0.047* ^(a)Extent of BrdU staining on the ipsilateral SVZ in cells per mm² ^(b)Extent of BrdU staining on the contralateral SVZ in cells per mm² ^(c)Lesion volume is expressed in percent compared to the contralateral section of the brain. ^(d)Synaptophysin, a marker for synapses, is expressed in % in IBZ ^(e)Apoptosis is expressed as the number of TUNEL+ cells per mm² ^(f)Total perimeter of 10 vWF positive vessels in a 40x microscopic field (μm) ^(g)Density of vWF vessels in IBZ (vessels/mm²) *indicates significant at p < 0.05

TABLE 23-11 Student's t-test comparisons of histology measures - IE route Cell A (hUTC) Cell B (PBS) Variable (N = 8) (N = 8) p-value BrdU Contralateral^(a)  196.2 ± 102.2 162.6 ± 52.3 0.421 BrdU Ipsilateral^(b)  552.7 ± 167.3 383.1 ± 84.0 0.023* Lesion volume^(c) 32.6 ± 6.4 32.4 ± 9.4 0.951 Synaptophysin^(d) 18.4 ± 3.7 14.8 ± 2.5 0.041* TUNEL (apoptosis)^(e)  32.5 ± 14.4  48.4 ± 12.5 0.033* vWF Perimeter^(f) 2482.7 ± 547.1 1745.1 ± 532.2 0.016* vWF vessel^(g) 245.8 ± 40.1 196.8 ± 27.6 0.013* ^(a)Extent of BrdU staining on the ipsilateral SVZ in cells per mm² ^(b)Extent of BrdU staining on the contralateral SVZ in cells per mm² ^(c)Lesion volume is expressed in percent compared to the contralateral section of the brain. ^(d)Synaptophysin, a marker for synapses, is expressed in % in IBZ ^(e)Apoptosis is expressed as the number of TUNEL+ cells per mm² ^(f)Total perimeter of 10 vWF positive vessels in a 40x microscopic field (μm) ^(g)Density of vWF vessels in IBZ (vessels/mm²) *indicates significant at p < 0.05

TABLE 23-12 Student's t-test comparisons of histology measures - REM route Cell A (hUTC) Cell B (PBS) Variable (N = 8) (N = 8) p-value BrdU Contralateral^(a) 186.1 ± 62.7 155.7 ± 60.3 0.339 BrdU Ipsilateral^(b)  469.3 ± 167.3 324.1 ± 76.9 0.043* Lesion volume^(c)  31.5 ± 10.4  31.3 ± 11.6 0.965 Synaptophysin^(d) 17.3 ± 3.9 13.2 ± 3.4 0.041* TUNEL (apoptosis)^(e)  30.2 ± 11.5  55.5 ± 36.0 0.079 vWF Perimeter^(f) 2546.0 ± 843.4 1909.2 ± 597.3 0.103 vWF vessel^(g) 250.6 ± 43.6 197.9 ± 32.2 0.016* ^(a)Extent of BrdU staining on the ipsilateral SVZ in cells per mm² ^(b)Extent of BrdU staining on the contralateral SVZ in cells per mm² ^(c)Lesion volume is expressed in percent compared to the contralateral section of the brain. ^(d)Synaptophysin, a marker for synapses, is expressed in % in IBZ ^(e)Apoptosis is expressed as the number of TUNEL+ cells per mm² ^(f)Total perimeter of 10 vWF positive vessels in a 40x microscopic field (μm) ^(g)Density of vWF vessels in IBZ (vessels/mm²) *indicates significant at p < 0.05

TABLE 23-13 Student's t-test comparisons of histology measures - IT route Cell A (hUTC) Cell B (PBS) Variable (N = 8) (N = 8) p-value BrdU Contralateral^(a) 199.8 ± 90.2  171.8 ± 113.7 0.594 BrdU Ipsilateral^(b)  493.8 ± 136.0  316.2 ± 120.6 0.015* Lesion volume^(c) 32.5 ± 8.0  31.0 ± 111 0.762 Synaptophysin^(d) 17.0 ± 3.9 12.9 ± 4.1 0.062 TUNEL (apoptosis)^(e)  23.4 ± 10.2  45.0 ± 26.9 0.051 vWF Perimeter^(f) 2254.2 ± 723.3 1879.1 ± 573.5 0.270 vWF vessel^(g) 233.5 ± 29.5 199.9 ± 19.2 0.017* ^(a)Extent of BrdU staining on the ipsilateral SVZ in cells per mm² ^(b)Extent of BrdU staining on the contralateral SVZ in cells per mm² ^(c)Lesion volume is expressed in percent compared to the contralateral section of the brain. ^(d)Synaptophysin, a marker for synapses, is expressed in % in IBZ ^(e)Apoptosis is expressed as the number of TUNEL+ cells per mm² ^(f)Total perimeter of 10 vWF positive vessels in a 40x microscopic field (μm) ^(g) Density of vWF vessels in IBZ (vessels/mm²) *indicates significant at p < 0.05

TABLE 23-14 Student's t-test comparisons of histology measures - IV route Cell A (hUTC) Cell B (PBS) Variable (N = 8) (N = 8) p-value BrdU Contralateral^(a) 222.6 ± 80.4 1511 ± 81.3  0.099 BrdU Ipsilateral^(b)  562.9 ± 117.7 377.6 ± 172.5 0.025* Lesion volume^(c)  32.3 ± 12.2 32.1 ± 10.8 0.983 Synaptophysin^(d) 18.4 ± 4.4 13.4 ± 3.5  0.026* TUNEL (apoptosis)^(e)  28.2 ± 17.5 55.8 ± 22.4 0.014* vWF Perimeter^(f) 2770.9 ± 610.6 2050.8 ± 695.6  0.045* vWF vessel^(g) 258.0 ± 45.9 210.8 ± 18.0  0.017* ^(a)Extent of BrdU staining on the ipsilateral SVZ in cells per mm² ^(b)Extent of BrdU staining on the contralateral SVZ in cells per mm² ^(c)Lesion volume is expressed in percent compared to the contralateral section of the brain. ^(d)Synaptophysin, a marker for synapses, is expressed in % in IBZ ^(e)Apoptosis is expressed as the number of TUNEL+ cells per mm² ^(f)Total perimeter of 10 vWF positive vessels in a 40x microscopic field (μm) ^(g) Density of vWF vessels in IBZ (vessels/mm²) *indicates significant at p < 0.05

hUTC treatment had no effect on cell proliferation in the contralateral hemisphere, or on lesion volume for all routes of administration. Administration of hUTC by all routes showed an increase in both cell proliferation and angiogenesis in the ipsilateral hemisphere when compared to PBS control cohorts. Apoptosis/cell death was decreased in those rats receiving hUTC through IC and IV routes. hUTC administered by all but intrathecal administration demonstrated an increase in synaptophysin expression when compared to PBS control. Consistent improvement across all histological parameters was observed in those cohorts receiving hUTC via IC and IV administration. These same routes were also associated with the most consistent significant functional improvement in the behavioral assays.

Tissue Distribution

Primer Specificity

Prior to sample analysis, primers utilized in this study were tested for specificity to human DNA transcripts. DNA isolated from rat astrocytes, U-87 cells (human glioblastoma-astrocytoma), mouse endothelial cells and hUTC were probed with human specific primers described above. The results (see Table 23-15) show that only human samples have detectable Ct values and that DNA isolated from two human sources yield similar data. Furthermore, gel electrophoresis revealed a PCR product of 135 bp (predicted size) only in DNA derived from human cells. These data confirm the species specificity of the primers utilized in this study.

TABLE 23-15 PCT confirms ERV-3 is species specific Rat Mouse HumanU87 Human hUTC Neg. Control Ct Ct Ct Ct Ct 0 0 27.8597 26.1838 0 0 0 26.829 270412 0 0 0 26.685 26.334 0 0 0 26.316 27.1845 0 0 0 26.6361 26.7741 0 0 26.5916 28.3466 Average 0 0 26.8195667   26.9773667 0

Optimal Concentration of DNA.

DNA isolated from hUTC was used to determine the optimal concentration of DNA to be employed in the PCRs. DNA was diluted from a 2.0 μg stock and tested in real time PCR reactions using the primers described. It was determined that DNA concentrations above 1.0 μg led to signal saturation, while concentrations below 1.0 μg generated inconsistent data (data not shown). Therefore, it was determined that 1.0 μg of DNA would be the optimal concentration and all stock samples were diluted as such.

Standard Curve

Genomic DNA isolated from samples of hUTC serially diluted (1:2) from 40,000 cells to ˜2 cells in autoclaved ddH₂0 was run via the previously described PCR method to generate a standard curve. Samples made from less than 46 cells resulted in undetectable levels of ERV-3 and were therefore eliminated from the standard curve. The following Ct values are reported and an inverse power function was used to generate a trendline (see Table 23-16; FIG. 15).

TABLE 23-16 PCR derived CT Values for Human Genomic DNA Standard Curve Generation Cell # Ct Value 39 39.31 78 39.07 156 38.49 312 37.05 625 35.79 1,250 34.76 2,500 34.18 5,000 33.67 10,000 32.27 20,000 31.22 40,000 30.77

In the brain, signal was detected in both hemispheres primarily for CM, IT and IC routes of administration. IA administration showed signal in some animals in the contralateral-left hemisphere, while after IV administration, only 2 animals exhibited cells in the contralateral hemisphere.

Serum Antigenicity Analysis—Presence of anti-hUTC IgG:

Blood was drawn before treatment as a baseline control and at Day 7, 14, 28, and 60 post-cell injection. Analysis of serum IgM levels did not show a clear indication of sensitization against hUTC in animals that received cell treatment at any of the time points (data not shown). All animals that received cells, regardless of the route of administration had detectable serum levels of IgG directed against activated UTC at every time point examined post-cell injection compared to PBS vehicle controls (see FIG. 16). This finding is consistent with xenogeneic cell delivery.

Conclusions

The present study was conducted to evaluate functional recovery and brain histology following administration of hUTC to male Wistar rats by different routes of administration: intravenous (IV), intraarterial (IA), intracisterna magna (ICM), intrathecal (IT), and intracerebral (IC). Functional recovery was assessed at various time points over a 60-day period and compared to PBS controls. Histology was performed on brain tissue to evaluate changes at the cellular level. Additionally, an arm of the study was conducted in cohorts of animals from which blood was drawn for the detection of antibodies against hUTC in serum at 7 days, 14 days, 28 days, and 60 days post-stroke in comparison to serum collected 1 day prior to cell administration (baseline). The same animals were also used to examine tissue distribution of hUTC using PCR.

Functional Recovery and Histology

Significant functional improvements in behavioral test scores in cell treatment groups were observed as early as Day 7 post insult. The intravenous route of administration demonstrated the most consistent functional recovery response attributable to the hUTC across all three behavioral tests. The intracerebral route was second in comparison. IA, ICM and IT routes of administration also demonstrated significant points of functional deficit reduction through the duration of the study, but not with the consistency demonstrated with IV or IC administration. Histological analysis showed hUTC treatment had no effect on cell proliferation in the contralateral hemisphere, or on lesion volume regardless of the route of administration. Administration of hUTC by all routes showed a significant increase (p<0.05) in cell proliferation and vessel number in the ipsilateral hemisphere. Vessel diameter was significantly increased (p<0.05) in IC and IV routes. Apoptosis and cell death was also significantly decreased (p<0.05) in rats receiving hUTC via IA, IC and IV routes. Additionally, hUTC administered by all but intrathecal administration demonstrated a significant increase (p<0.05) in synaptophysin expression over PBS treated controls. In summary, consistent improvement across all histological parameters was observed in IC and IV hUTC administration cohorts. It is noted that these same routes were associated with the most consistent improvement in behavioral assays over the 60-day testing period.

Tissue Distribution

To gain further understanding on the persistence and distribution of hUTC, a PCR based method was employed to detect human cells in rat organs by their genomic DNA (see Lee, S-T. et al. 2006. Journal of Neuroscience Methods 157:225-229). The collected data were inconclusive and showed that, with the exception of the brain, cells were not detected in organs harvested from animals 60 days post treatment. High, inconsistent and broad ranges in Ct values were reported. There are several explanations for these results. First, hUTC might not persist in measurable amounts in rat tissue at time points as late as 60 days post-administration. In addition, the high Ct values reported for the samples used to make the standard curve may suggest assay sensitivity issues. Therefore, this assay may need further optimization.

Antigenicity

The elevated mean fluorescence intensity (MFI) observed in the serum antibody FACS assay in samples derived from cell treatment groups is indicative of the (rat-anti-human IgG) antibody titer in serum. The MFI across the various routes of administration used in the MCAo model were much higher overall than values observed in other studies involving allogeneic inflammatory models and xenogeneic non-inflammatory models.

Overall, a significant reduction (p<0.05) in behavioral deficits was observed in all cell treatment cohorts with IV administration demonstrating the most robust effects. The IC route was the second most consistent. IA, ICM and IT routes of administration also demonstrated significant points of functional recovery but not with the consistency demonstrated with IV or IC administration. The histology showed significant increases (p<0.05) in cell proliferation, vessel number and for all routes of administration. The diameter of microvessels was significantly increased (p<0.05) in IC and IV routes. Apoptosis was significantly decreased (p<0.05) in IA, IC and IV delivery routes only, while synaptophysin expression was significantly (p<0.05) increased in all but IT delivery routes. Efforts to detect hUTC via human genomic DNA detection methods proved inconclusive. Finally, the production of antibodies (IgG) to hUTC was confirmed in this xenogeneic experiment for all delivery routes; nevertheless, a significant increase in functional recovery observed in the cell treatment cohorts.

References for Example 23

-   Institute of Laboratory Animal Resources, Guide for the Care and Use     of Laboratory Animals, 7th edition. National Academy Press,     Washington, D.C., 1996. -   Chen H. et al. 1992. The effect of hypothermia on transient middle     cerebral artery occlusion in the rat. J Cereb Blood Flow Metab     12:621-8. -   Seyfried D M et al. 2008 Mannitol enhances delivery of marrow     stromal cells to the brain after experimental intracerebral     hemorrhage. Brain Res. 1224: 12-9. -   Zhang Z G et al. 2003. Magnetic resonance imaging and neurosphere     therapy of stroke in rat. Ann Neurol. 53(2):259-63. -   Li Y et al. 2000. Intrastriatal transplantation of bone marrow     nonhematopoietic cells improves functional recovery after stroke in     adult mice. J Cereb Blood Flow Metab. (9):1311-9. -   Chen J et al. 2000. Intracerebral transplantation of bone marrow     with BDNF after MCAo in rat. Neuropharmacology. 39(5):711-6. -   Lepore A C et al. 2005. Neural precursor cells can be delivered into     the injured cervical spinal cord by intrathecal injection at the     lumbar cord. Brain Res. 1045(1-2):206-16. -   Chen, J. et al. 2001. Therapeutic benefit of intravenous     administration of bone marrow stromal cells after cerebral ischemia     in rats. Stroke 32:1005-11 -   Zhang, L. et al. 2002a. A test for detecting long-term sensorimotor     dysfunction in the mouse after focal cerebral ischemia. J Neurosci     Methods 117:207-14. -   Schallert, T. et al. 1997. Use-dependent structural events in     recovery of function. Adv Neurol 73:229-38. -   Paxinos, G., and C. Watson. 1986. The rat brain in stereotaxic     coordinates, 2nd edition. Sydney; Orlando: Academic Press. -   Swanson, R. A. et al. 1990. A semi-automated method for measuring     brain infarct volume. J Cereb Blood Flow Metab 10:290-3. -   Chen J. et al. 2004. Combination therapy of stroke in rats with a     nitric oxide donor and human bone marrow stromal cells enhances     angiogenesis and neurogenesis. Brain Res. 105:21-8. -   Chen J. et al. 2003. Statins induce angiogenesis, neurogenesis, and     synaptogenesis after stroke. Ann. Neurol. 53:743-51. -   Li, Y. et al 1998. Intact, injured, necrotic and apoptotic cells     after focal cerebral ischemia in the rat. J Neurol Sci 156: 119-32. -   Li, Y. et al. 1995. Ultrastructural and light microscopic evidence     of apoptosis after middle cerebral artery occlusion in the rat. Am J     Pathol 146: 1045-51. -   Kollmar, R. et al. 2002. Neuroprotective effect of delayed moderate     hypothermia after focal cerebral ischemia: an MRI study. Stroke     33:1899-1904. -   Matsuo, Y. et al. 2001. Protective effect of endothelin type A     receptor antagonist on brain edema and injury after transient middle     cerebral artery occlusion in rats. Stroke 32:2143-2148. -   Mokudai, T. et al. 2000. Delayed treatment with nicotinamide     (Vitamin B3) improves neurological outcome and reduces infarct     volume after transient focal cerebral ischemia in Wi star rats.     Stroke 31:1679-1685. -   Pfefferkorn T, and Rosenberg G A. 2003. Closure of the blood-brain     barrier by matrix metalloproteinase inhibition reduces rtP     A-mediated mortality in cerebral ischemia with delayed reperfusion.     Stroke 34:2025-2030. -   Lee, S-T. et al. 2006. Quantification of human neural stem cell     engraftments in rat brains using ERV-3 real-time PCR. Journal of     Neuroscience Methods 157: 225-229.

Example 24 hUTC in a Rat Transient Middle Cerebral Artery Occlusion (MCAo) Stroke Model: Intra Cisterna Manna Time-of-Administration and Dose-Response Sturdy

A previous study demonstrated that intra cisterna magna (ICM) administration of hUTC significantly improved neurological functional recovery when administered 1 day after MCAo. The study in this example evaluated the dose responses and the therapeutic window of intra cisterna magna administration of hUTC in rats subjected to 2 hours transient MCAo. In addition, the effects of hUTC in rats subjected to 2 hours of MCAo in the presence and absence of an immunosuppressive agent, cyclosporine (CsA), were evaluated.

Methods

Ischemic Stroke Model in Rat:

Male Wistar rats (2 to 3 months old) weighing 270 to 300 grams were employed in the experiments. Transient focal cerebral ischemia was induced using a method of intraluminal filament occlusion of the middle cerebral artery, as previously described (Chen, H. et al. 1992. J. Cereb Blood Flow Meta 12:621-9). Briefly, rats were anesthetized with 3.5% isoflurane in N₂O:O₂ (2:1) and maintained at 1%-1.5% isoflurane using a facemask. The right common carotid artery, external carotid artery and internal carotid artery were exposed. A length of 4-0 monofilament nylon suture (18.5-19.5 mm), determined by the animal weight, with its tip rounded by heating near a flame, was advanced from the external carotid artery into the lumen of the internal carotid artery until it blocked the origin of the MCA. Two hours after MCAo, reperfusion was performed by withdrawal of the suture until the tip cleared the internal carotid artery.

Cell Transplantation:

Animals were anesthetized with 3.5% isoflurane in N2O:O2 (2:1) and maintained at 0.5-1.5% isoflurane using a facemask. 96 rats were randomized into one of the following nine groups:

1. hUTC at a dose of 1 million/40 μl/rats at 24 hours after MCAo 2. Vehicle (CSCV4) at a dose of 40 μl/rats at 24 hours after MCAo 3. hUTC at a dose of 1 million/40 μl/rats at 15 days after MCAo 4. Vehicle at a dose of 40 μl/rats at 15 days after MCAo. 5. hUTC at a dose of 1 million/40 μl/rats at 30 days after MCAo 6. hUTC at a dose of 0.3 million/40 μl/rats at 30 days after MCAo 7. Vehicle at a dose of 40 μl/rats at 30 days after MCAo 8. hUTC with cyclosporine (CsA) at a dose of 1 million/40 μl/rats at 24 hours after MCAo 9. Vehicle with CsA at a dose of 40 μl/rats at 24 hours after MCAo For ICM injection, rats were placed on ear bars of a Kopf stereotaxic frame and the cisterna magna was penetrated with a 22-gauge Hamilton syringe. After confirming the correct positioning of the needle into the cisterna magna by the reflux of cerebrospinal fluid (CSF), hUTC at a dose of 1×10⁶/40 μl or the same volume of vehicle were injected over 5 seconds.

For histological studies, rats (n=8/group) were allowed to survive 60 days after cell administration. Rats treated with hUTC with or without CsA at 1 day after stroke, or treated with hUTC only at 30 days after stroke, were sacrificed at 1 hour or 24 hours after cell injection (groups 1, 5, and 8; n=3/group) for major organ and blood collection. In addition, serum samples were collected on hUTC treated rats with or without CsA (groups 1 and 8, n=3/group) immediately prior, and 1, 7, 28, 42, 60 days after cell injection.

Serum samples were evaluated as were other samples including cerebral (left and right), spinal cord (cervical, thoracic, and lumber), heart, lungs, liver, spleen, kidney (left and right), testis (left and right), CSF, and whole blood.

Cyclosporine (CsA) injection (IP):

Cyclosporine (CsA) at a dose of 4 mg/kg was intraperitoneally administered to rats daily for 14 days starting at the time of MCAo. Thereafter, cyclosporine was administered twice per week for the remainder of the studies.

Neurological Functional Tests:

A battery of behavioral tests were measured before MCAo (baseline) and weekly after MCAo starting at 1 day after MCAo (n=8/group) by an investigator who was blinded to the experimental groups.

Modified neurological severity score (mNSS) (Chen, J. et al. 2001 Stroke 32:1005-11): mNSS is a composite of motor, sensory, balance and reflex tests. Neurological function was graded on a scale of 0 to 18 (normal score 0; maximal deficit score 18). In the severity scores of injury, one point is awarded for the exhibition of a specified abnormal behavior or for the lack of a tested reflex. Thus, the higher score, the more severe is the injury.

Adhesive-removal patch test (Schallert, T. et al. 1997 Adv Neurol. 73:229-38): Two small pieces of adhesive-backed paper dots of equal size (113.1 mm²) were used as bilateral tactile stimuli occupying the distal-radial region of each forelimb. The time to remove each stimulus from forelimbs was recorded on three trials per day. Individual trials were separated by at least 5 minutes. Before surgery, the animals were trained for 3 days. Once the rats were able to remove the dots within 10 seconds, they were subjected to MCAo. To increase the level of difficulty for the test, the size of the adhesive dots was reduced in half starting from 2 months after stroke (groups 3 to 7).

Foot-fault test (Zhang, L. et al. 2002a J. Neurosci Methods 117:207-14): Rats were tested with the Foot-fault test for placement dysfunction of the forelimb. Rats were placed on elevated hexagonal grids of different sizes. Rats placed their paws on the wire while moving along the grids. With each weight-bearing step, the paw might fall or slip between the wire. This was recorded as a foot-fault. The total number of steps (movement of each forelimb) used to cross the grid was counted up to 100 steps or ten minutes, whichever came first, and the total number of Foot-faults for each forelimb were recorded. Data are presented as the percentage of contralateral Foot-faults per total steps.

Brain Section Preparation:

Rat brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde. Using a rat brain matrix (Activational Systems Inc., Warren, Mich.), each forebrain was cut into 2 mm thick coronal blocks for a total 7 blocks from bregma 5.2 mm to bregma −8.8 mm per animal (Paxinos and Watson 1986). The tissues were embedded in paraffin and a series of 6 μm-thick slides were cut.

Measurement of Infarct Area:

One of each coronal paraffin slides (6 μm thick) from seven blocks was stained with hematoxylin and eosin (H&E). The seven brain slides were traced using the Global Lab Image analysis system (Data Translation, Marlboro, Mass.). The indirect lesion area, in which the intact area of the ipsilateral hemisphere was subtracted from the area of the contralateral hemisphere, was calculated (Swanson, R. et al. 1990. J Cereb Blood Flow Metab 10:290-3). The lesion volume is presented as a volume percentage of the lesion compared to the contralateral hemisphere.

Immunohistochemistry:

A standard paraffin block was obtained from the center of the lesion, corresponding to coronal coordinates for bregma between approximately −1.0 and 1.0 mm. A series of 6-μm-thick sections were cut from this block and were analyzed by light and fluorescent microscopy (Olympus, BH-2).

Von Willebrand factor (vWF for endothelial cells, 1:400, Dako) and synaptophysin (1:500, Chemicon) antibodies were used to identify vessels and synapses, respectively.

For measurement of VVF immunoreactive vessels and synaptophysin, 8 fields of view from the ischemic boundary zone (IBZ, FIG. 5) was digitized using a 40× objective via the MicroComputer Imaging Device (MCID) analysis system. The numbers of vessels and the positive area of synaptophysin in the IBZ were evaluated throughout each field of view. Vascular perimeter was also measured and presented as the total perimeter of 10 enlarged vessels in the ipsilateral brain.

Analysis

The study tested hUTC dose-response, treatment window, and the effect of CsA on hUTC mediated functional recovery, measured by three behavioral tests at day 60 after the treatment onset as the primary outcome. The functional recovery at the other days and histological evaluation measurements were considered as the secondary endpoints. Data were evaluated for normality. Data transformation was considered if data were not normal. As the result, the distribution of the behavioral data was not normal. Therefore, ranked data for behavior tests were used for the analysis.

The global test using Generalize Estimating Equation (GEE) was implemented to test the group difference on functional recovery measured from three behavioral tests. The analysis started testing the two-treatment combination effect (interaction) or overall treatment effect, followed by testing the subgroup analysis, if the interaction or main effect was detected at the 0.05 level. Any sub-group analysis was considered as exploratory if there is no overall group effect or the treatment interaction at the 0.05 level. The global test on multiple outcomes is more efficient than a single outcome, when the group effects are consistent on all the outcomes (e.g., the positive correlation).

The analyses had three components and animals were shared: (1) Component 1: study hUTC and CsA combination effect with treatment window at day 1 after the stroke onset; (2) Component 2: study hUTC administration windows; and (3) Component 3: study hUTC dose (0, 0.3 and 1 M) effect administered at day 30 of stroke onset.

Results

Mortality

102 animals were employed for MCAo in this experiment. Five rats died within 1 day after MCAo before initiation of treatment due to stroke complications such as subarachnoid hemorrhage and brain edema. One rat from group 3 died immediately after cell injection due to ICM injection associated intracranial hemorrhage.

Neurological Functional Outcomes

hUTC with or without CsA at day 1. Treatment with hUTC at 1 day after MCAo with or without CsA significantly reduced neurological functional deficits at day 14, 28, 35, 42, 49, 56, and 60 after stroke compared with rats treated with vehicle (see FIG. 17). Treatment with CsA did not improve the neurological functional outcome compared to the vehicle control. The combination of hUTC with CsA significantly reduced neurological functional deficits compared to rats treated with hUTC alone at day 28, 35 and 56 after stroke. In FIG. 17, significant improvement is indicated by *.

hUTC administered at 15 days after MCAo. Treatment with hUTC (1×10⁶) at 15 days after MCAo significantly improved neurological functional deficits starting at day 42 and persisted up to day 75 after stroke compared with vehicle treated rats (see FIG. 18). In FIG. 18, significant improvement is indicated by *.

hUTC administered at 30 days after MCAo. Treatment with hUTC (1×10⁶) at 30 days after MCAo significantly improved neurological functional deficits at day 49, 63, 70, 77, 84, and 90 after stroke compared with vehicle treated rats (see FIG. 19). In FIG. 19, significant improvement is indicated by *.

hUTC at doses of 3×10⁵ and 1×10⁶ administered 30 days after MCAo. For rats receiving a dose of 1×10⁶ hUTC at 30 days after MCAo, significant functional improvements were observed starting at day 42 and persisted up to day 90 post stroke. For rats receiving a dose of 3×10⁵ hUTC at 30 days after MCAo, significant functional improvements were observed at day 49, 63, 70, 77. 84, and 90 after stroke compared with vehicle treated rats (see FIG. 20). In FIG. 20, significant improvement is indicated by *.

Body Weight. As indicated in FIG. 21, no significant differences in body weight were detected among the groups.

Histology

Lesion Volume. As indicated in FIG. 22, no significant differences of lesion volumes were detected among groups.

Vessel Density and perimeter in the IBZ. Treatment with hUTC with or without CsA at 1 day significantly increased the vWF immunoreactive vessel density in the IBZ compared with vehicle treated rats at 60 days after stroke (see FIG. 23 for vessel density). However, treatment with hUTC failed to increase the vWF immunoreactive vessel density in the IBZ compared with vehicle treated rats when administered at 15 or 30 days after stroke. No significant differences of vWF immunoreactive vessel perimeter were observed among groups.

Synaptophysin immunoreactivity in the IBZ. Treatment with hUTC with or without CsA significantly increased the percentage of synaptophysin immunoreactive area in the IBZ compared with vehicle treated rats when administered at 1 day after stroke. In addition, rats treated with hUTC at 15 days after stroke onset significantly increased the percentage of synaptophysin immunoreactive area in the IBZ compared with vehicle treated rats. However, treatment with hUTC failed to increase the percentage of synaptophysin immunoreactive area in the IBZ compared with vehicle treated rats when administered at 30 days after stroke onset.

Conclusion

All rats exhibited severe neurological functional deficits 24 hours after stroke with no significant differences among the groups. All rats exhibited the same level of neurological functional deficits 24 hours prior to treatment with no significant differences among the groups.

Rats receiving hUTC with or without CsA at 1 day showed significant improvement in neurological function at day 14, 28, 35, 42, 49, 56, and 60 compared with rats treated with vehicle. Treatment with CsA alone did not improve the neurological functional outcome compared to the vehicle control. Combination of hUTC with CsA significantly reduced neurological functional deficits at day 28, 35 and 56, but not at day 42, 49, and 60 compared to rats treated with hUTC alone, suggesting that administration of CsA did not enhance the treatment effects of hUTC on neurological functional recovery.

Treatment with hUTC at 15 days after stroke onset significantly improved neurological function starting at day 42 and persisted up to day 75 after stroke compared with vehicle treated rats.

Treatment with hUTC (1×10⁶) at 30 days after stroke onset significantly improved neurological function at day 49, 63, 70, 77, 84, and 90 compared to their matched controls.

Example 25 Efficacy of Intranasal Delivery of hUTC on Functional Recovery Following Permanent Middle Cerebral Artery Occlusion (pMCAo) in Rats

Previous studies have shown that treatment with hUTC can improve neurological recovery in rats that were subjected to either tMCAo or pMCAo. This has been demonstrated using a variety of cell doses and delivery methods. Thus far, the delivery methods employed have been intravenous (IV), intra-arterial (IA), intrathecal (IT), intracisternal magna (ICM), and intra cerebral (IC). This example evaluated the feasibility and efficacy of intranasal delivery of hUTC in rodent model of ischemic stroke. Two groups of rats were pretreated with hyaluronidase followed by delivery of one (1) million hUTC or vehicle control 24 hours post-pMCAo. Behavioral assessments of motorsensory functions, including limb placing, body swing test were assessed.

Materials and Method:

Cell Preparation:

hUTC were expanded from a working cell bank (Lonza, lot 0000106053, vial 98) to approximately thirty (30) population doublings on Hillex microcarrier beads and cyropreserved in CSCV 4. Cells were shipped to the test location in an arctic express dry shipper at temperatures ≦−120° C. and placed into liquid nitrogen storage upon arrival.

Prior to cell shipment, endotoxin, mycoplasma, viability, total number of cells per vial, population doublings, karyology and protein expression were determined for each lot of cells and found to be within specification.

On days of cell administration, vials were thawed and prepared for administration. In brief, cells were thawed in a 37° C. water bath and counted. Cells were kept on ice prior to administration and viability determined by Trypan blue staining Cells were suspended in sufficient CSCV4 to provide administration of 1 million cells in a 48 μl preparation.

Animals, Housing and Diet:

Twenty-four (24) male Sprague Dawley Rats between 225 to 275 g in weight were ordered 7 to 14 days prior to surgery (Charles River Laboratories, Kingston, N.Y.). Rats were approximately 300 to 400 grams at study initiation. This species was selected to utilize an established method of inducing permanent cerebral ischemic stroke.

The animals utilized in this study were handled and maintained in accordance with the current requirements of the Animal Welfare Act. Compliance was accomplished by adhering to the Animal Welfare regulations (9 CFR) and conforming to the current standards promulgated in the Guide for the Care and Use of Laboratory Animals.

Animals were housed two per cage in ventilated solid bottom cages with Bed-O-Cobs® used for bedding, unless severe aggression or injury was displayed in which case animals were housed singly. Animal rooms were provided with filtered air at a temperature of 21±2° C. and 50%±20% relative humidity. The room had on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight and animals were fed with standard Purina 5001 chow. Food and water was provided ad libitum. Animal care was provided according to approved institutional protocols and guidelines.

Study Design

The experimental design was as outlined in Table 25-1 below:

TABLE 25-1 Experimental Design Handle the Animals for 7 days before the surgery  7 days Cefazolin Sodium i.p. (40 mg/kg), right before surgery Buprenorphine s.c. (0.1 mg/kg), right before surgery PMCAo (Day 0, modified Tamura model)  1 day Animals receive cells, or vehicle: intranasally, at Day 1 after PMCAo 42 days Animals are sacrificed on Day 42 after PMCAo Behavioral Evaluation: Day −1 (pre-operation), Day 1, Day 7, Day 14, Day 21, Day 28, and Day 41: On days when both behavioral test and treatments were done on the same animals, the behavioral testing will be done prior to treatments. Investigators blinded to treatment assignment did the behavioral evaluations.

Animal Preparation and allocation of treatment groups

Twenty-four (24), adult, male Sprague-Dawley rats as described above were used for the study (including 4 extra animals). All rats were housed and handled for behavioral assessment for seven (7) days prior to surgery for acclimation purposes. At the end of the handling period, rats were randomized and assigned to different groups.

The amount of time needed for some procedures in this study necessitated breaking up the two (2) treatment groups (as listed below), into four (4) working groups. Five animals received stroke surgery per day. If an animal died during the 4-day surgical period of the study, it was replaced by a spare. If not, the animal was not replaced (due to surgical and behavioral schedules).

TABLE 25-2 Allocation of Animals to Treatment Group Treatment # of rats Day of Sacrifice 1 CSC4 10 42 2 hUTC (1 × 10⁶ cells) 10 42 Both vehicle and cell treatments were administered intranasally in an identical manner in a volume of 48 μl.

Surgical Preparation: Middle Cerebral Artery Occlusion (MCAo) (Tamura Model)

The model for cerebral stroke to be employed in this study was a permanent occlusion of the proximal right middle cerebral artery using a modification of the method first described by Tamura et al. (Carmichael T. 2005. Rodent Model of Focal Stroke 2:396-409; Koizumi, J. et al. 1986. Jpn J Stroke 8:108; Tamura, A. et al. 1981. J Cereb Blood Flow Met 1:53-60; Chen, S T. et al. 1986. Stroke 17:738-743).

Male Sprague-Dawley rats (300 to 400 g at the time of surgery) were anesthetized with 2-3% isofluorane in the mixture of N₂O:O₂ (2:1), and were maintained with 1-1.5% isoflurane in the mixture of N₂O:O₂ (2:1). The temporalis muscle was bisected and reflected through an incision made midway between the eye and the eardrum canal. The proximal MCA was exposed through a subtemporal craniectomy without removing the zygomatic arch and without transecting the facial nerve. The artery was then occluded by microbipolar coagulation from just proximal to the olfactory tract to the inferior cerebral vein, and was transected. Body temperature was maintained at 37.0±1° C. throughout the entire procedure. Cefazolin (40 mg/kg; West-Ward, Lot 091011.1; Exp. January 2012) was given intraperitoneally (i.p.) before MCAO to prevent infections. Buprenorphine (Bedford Labs, 1641650, Exp. September 2010; 0.05˜0.1 mg/kg) was given subcutaneously (s.c.) before the MCAO surgery as analgesia.

Test Article Administration

Cells (hUTC, 1×10⁶ in 48 μl) or vehicle (48 μl) were administered following MCAo. Animals were anesthetized as above (1-3% isoflurane in N₂O:O₂ (2:1)). Rats were placed on their back on a heating pad with a suitable cover. A rolled up 2×2 gauze pad, held together with tape, was placed under the rats neck, thus keeping the neck of the rat flat and horizontal.

Hyaluronidase Pretreatment

Twenty-four (24) microliters of hyaluronidase (HU) (Sigma H3756, Lot: 049K8724, stored at −20° C., 200 U in PBS w/o Ca/Mg) were administered as four 6 μl drops delivered using a 10-20 μl pipette with a sterile tip. Animals received one drop every two minutes to alternating nares of the nose (left, right, left, right), gently covering/occluding one naris to place the 6 μl drop on the opening of the other naris. Animals were left on their backs in the delivery position for an additional ten minutes after the last drop of hyaluronidase was delivered, before being allowed to recover from anesthesia. Twenty minutes later, the animal was re-anesthetized for the delivery of test article.

Treatment Delivery

Animals were re-anesthetized as above. One million hUTC suspended in a total volume of 48 μl of CSCV4, were administered as eight 6 μl drops delivered using a 10-20 μl pipette with a sterile tip. Animals received one drop every two minutes to alternating nares of the nose, gently covering/occluding one naris to place the 6-μl drop on the opening of the other naris. Animals were left on their backs in the delivery position for an additional ten minutes after the last drop of hUTC was delivered, before being allowed to recover from anesthesia. Control animals received 48 μl of CSCV4 without hUTC using the identical procedure. hUTC was kept on ice between doses.

Neurological Functional Tests

Functional activities were evaluated using limb placing and body swing behavioral tests. These tests were performed one day prior to surgery (Day −1), one day after surgery (before treatments) (Day 1) and at seven (Day 7), fourteen (Day 14), twenty-one (Day 21), twenty-eight (Day 28) and forty-one days (Day 41) after MCAo by an investigator who was blinded to the identity of the experimental groups.

Limb Placing Test

The limb placing tests are divided into both forelimb and hindlimb tests. For the forelimb-placing test, the examiner holds the rat close to a tabletop and scores the rat's ability to place the forelimb on the tabletop in response to whisker, visual, tactile, or proprioceptive stimulation. Similarly, for the hindlimb placing test, the examiner assesses the rat's ability to place the hindlimb on the tabletop in response to tactile and proprioceptive stimulation. Separate sub-scores are obtained for each mode of sensory input and added to give total scores (for the forelimb placing test: 0=normal, 12=maximally impaired; for the hindlimb placing test: 0=normal; 6=maximally impaired).

Scores are given in half-point increments (see Table 25-3 below). Typically, there is a slow and steady recovery of limb placing behavior during the first month after stroke.

TABLE 25-3 Limb Placing Scoring Forelimb placing test (0-12): Whisker pacer (0-2) Visual placing (forward (0-2), sideways (0-2)) Tactile placing (dorsal (0-2), lateral (0-2)) Proprioceptive placing (0-2) Hindlimb placing test (0-6): Tactile placing (dorsal (0-2), lateral (0-2)) Proprioceptive placing (0-2) For each subtest, animals 0.0 = immediate response are scored as follows: 0.5 = response within 2 seconds 1.0 = response of 2-3 seconds 1.5 = response of >3 seconds 2.0 = no response

Body Swing Test

The rat is held approximately one (1) inch from the base of its tail. It is then elevated to an inch above a surface of a table. The rat is held in the vertical axis, defined as no more than 10° to either the left or the right side. A swing is recorded whenever the rat moves its head out of the vertical axis to either side. Before attempting another swing, the rat must return to the vertical position for the next swing to be counted. Thirty (30) total swings are counted. A normal rat typically has an equal number of swings to either side. Following focal ischemia, the rat tends to swing to the contralateral side (left side in this case). Body swing scores are expressed as a percentage of rightward over total swings. There is a spontaneous partial recovery of body swing scores (toward 50%) during the first month after stroke.

Study Termination

At study termination, at forty-two days after MCAo, rats were anesthetized deeply with ketamine (50-100 mg/kg) and xylazine (5-10 mg/kg), mixture and exsanguinated under deep anesthesia. Rats were perfused transcardially with normal saline containing heparin (2 units/ml), followed by 4% paraformaldehyde for further histology analysis. Brains (including the olfactory bulbs) were harvested and post-fixed in 4% paraformaldehyde.

Data Analysis

All data are expressed as mean±S.E.M. Behavioral and body weight data were analyzed by repeated measures of ANOVA (treatment X time). Positive p values for the F-statistic for overall ANOVAs including all groups enabled pair wise ANOVAs between groups.

For the behavioral tests, the day before treatment (stroke), day −1 and the day of/after treatment (stroke), day 1, were purposely not included in the analysis to ensure normal distribution of the data.

Results:

Clinical Observation and Survival: All animals appeared to be normal.

Neurological Functional tests: FIGS. 25 to 27 show the results of the functional tests. As is evident from FIG. 25 (Forelimb Placing), although there were no significant differences, there was a trend toward enhanced recovery in hUTC-treated animals. Furthermore, as is evident from FIG. 26 (hindlimb placing), animals receiving hUTC were superior to vehicle group after stroke (p<0.01). In addition, as is evident from FIG. 27 (body swing), animals receiving hUTC were superior to vehicle group after stroke (p<0.001).

Weight Changes: No significant differences between the two groups were observed.

Conclusions:

Permanent right MCAo by the Tamura method was made in mature male Sprague-Dawley rats, resulting in focal right cerebral infarction. Human UTCs (1 MM) were administered intranasally at 1 day after MCAo, compared to appropriate controls. Behavioral assessments of sensorimotor function were made at 1 day before MCAo, 1 day after MCAo, and at 7, 14, 21, 28 and 41 days after MCAo. Human UTCs, given at 1 day after stroke, enhanced sensorimotor recovery compared to respective vehicle-treated controls on behavioral tests, especially hindlimb placing and body swing tests. In this study, it was demonstrated that hUTC can improve neurological recovery in rats when delivered intranasally following pretreatment with hyaluronidase. The efficacy with this mode of delivery is comparable to that of IV administration.

References for Example 25

-   Rodent Models of Focal Stroke: Size, Mechanism, and Purpose. S.     Thomas Carmichael Vol 2, 396-409, July 2005. -   Koizumi, J et al. 1986. Experimental studies of ischemic brain     edema. 1. A new experimental model of cerebral embolism in which     recirculation can be introduced into the ischemic area. Jpn J Stroke     8: 108. -   Tamura, A. et al. 1981. Focal cerebral ischemia in the rat: 1.     Description of technique and early neuropathological consequences     following middle cerebral artery occlusion. J Cereb Blood Flow Met     1: 53-60. -   Chen, S T. et al. 1986. A model offocal ischemic stroke in the rat:     reproducible extensive cortical infarction. Stroke 17: 738-743.

Example 26 Efficacy of Intranasal Delivery of hUTC With or Without Hyaluronidase on Functional Recovery Following Permanent Middle Cerebral Artery Occlusion (pMCAo) in Rats

This example evaluated the efficacy of intranasal delivery of hUTC in a rodent model of ischemic stroke with and without hyaluronidase (HU) pretreatment. Behavioral assessments of sensorimotor functions, including limb placing and body swing test were assessed. Distribution of hUTC in the nasal cavity, brain, lung, and blood is also be examined using rtPCR at 4 hours, 24 hours, 3 days and 41 days post delivery.

Experimental Groups were as follows:

-   1. 1 MM cells Intranasal with HU, Day 1 post-MCAo, sacrifice Day 1     (4 hours after dose) (N=4 for RNA) -   2. 1 MM cells Intranasal without HU, Day 1 post-MCAo, sacrifice Day     1 (4 hours after dose) (N=4 for RNA) -   3. 1 MM cells Intranasal with HU, Day 1 post-MCAo, sacrifice Day 2     (1 day after dose) (N=4 for RNA) -   4. 1 MM cells Intranasal without HU, Day 1 post-MCAo, sacrifice Day     2 (1 day after dose) (N=4 for RNA) -   5. 1 MM cells Intranasal with HU, Day 1 post-MCAo, sacrifice Day 4     (3 days after dose) (N=4 for RNA) -   6. 1 MM cells Intranasal without HU, Day 1 post-MCAo, sacrifice Day     4 (3 days after dose) (N=4 for RNA) -   7. 1 MM cells Intranasal with HU, Day 1 post-MCAo, sacrifice Day 42     (41 days after dose) (N=10 total; N=4 each from cell groups for RNA) -   8. 1 MM cells Intranasal without HU, Day 1 post-MCAo, sacrifice Day     42 (41 days after dose) (N=10 total; N=4 each from cell groups for     RNA) -   9. Vehicle Intranasal with HU, Day 1 post-MCAo, sacrifice Day 42 (41     days after dose) (N=10) -   10. Vehicle Intranasal without HU, Day 1 post-MCAo, sacrifice Day 42     (41 days after dose) (N=10)     Groups 7-10 above (N=10 animals/group) were subjected to behavioral     evaluation. Tissue for subsequent analysis was taken from Groups 1-8     above (N=4 animals/group).

Materials and Methods:

Animals, Housing and Diet:

Seventy-two (72) Male Sprague-Dawley rats, 250 to 275 g in weight, were ordered 7 to 10 days prior to surgery (Charles River Laboratories). They were allowed free access to food and water. Animals were assigned sequential identification numbers using permanent marker on the tail. The animals were observed the day prior to surgery, and those appearing to be in poor health were excluded from the study.

Animals were housed in rooms provided with filtered air at a temperature of 21±2° C. and 50%±20% relative humidity. The room was on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight. Shepherd's® ¼″ premium corn cob was used for bedding and 1 Nylabone® (3.5″, Dura bones Petite) and a Rat Runnel™ (BioServ K3326) was put in each cage. Animals were fed with Lab Diet® 5001 chow. Water was provided ad libitum.

The animals were housed two (2) per cage before and after surgery, unless severe aggression or injury was displayed, or death of cage mate, in which case animals were housed singly.

Study Design:

Animal Preparation:

Seventy-two (72), adult, male Sprague-Dawley rats as described above were used for the study (including 8 extra animals). All rats were housed for acclimation purposes. Animals that were assessed for behavior were handled for seven (7) days prior to surgery. At the end of the acclimation/handling period, rats were randomized and assigned to treatment groups.

Surgical Preparation: Middle Cerebral Artery Occlusion (MCAo), Tamura Model

Focal cerebral infarcts were made by permanent occlusion of the proximal right middle cerebral artery (MCA) using a modification of the method of Tamura et al. Male Sprague-Dawley rats (300 to 400 g at the time of surgery) were anesthetized with 2-3% isoflurane in the mixture of N₂O:O₂ (2:1), and were maintained with 1-1.5% isoflurane in the mixture of N₂O:O₂ (2:1). The temporalis muscle was bisected and reflected through an incision made midway between the eye and the eardrum canal. The proximal MCA was exposed through a subtemporal craniectomy without removing the zygomatic arch and without transecting the facial nerve. The artery was then occluded by microbipolar coagulation from just proximal to the olfactory tract to the inferior cerebral vein, and was transected. Body temperature was maintained at 37.0±1° C. throughout the entire procedure. Cefazolin (40 mg/kg; West-Ward, Lot 091011.1, Exp. 31 Jan 12; Hospira, Lot 101A012, Exp. 31 May 12) was given intraperitoneally (i.p.) before MCAo to prevent infections. Buprenorphine (0.05˜0.1 mg/kg; Reckitt Benckiser, Buprenex® Lot CN002701, Exp. 1 Oct. 12; Bedford Labs, Lot 1865303, Exp. 30 Jun. 11) was given subcutaneously (s.c.) before the MCAo surgery as analgesia.

Cell Preparation:

Cells (hUTC) and Vehicle (CSCV4) were sent by dry shipper and stored in liquid nitrogen. Cells were thawed each day of injection as per the protocol.

Dosing:

Cells (hUTC, 1 MM in 48 μl) or vehicle (48 μl) were administered through both nares on day 1 following MCAo. For this procedure, animals were placed under anesthesia (1˜3% isoflurane in N₂O:O₂ (2:1)) and were placed on their backs on a heating pad with a suitable cover. A rolled up 2×2 gauze pad, held together with tape, was placed under the rats neck, thus keeping the neck of the animal flat and horizontal. For the groups without HU, the pretreatment steps below were omitted.

Hyaluronidase Pretreatment:

Twenty-four (24) microliters of HU (Sigma H3756 Lot: 049K8724, stored at −20° C., 200 U in PBS w/o Ca/Mg) were administered as four 6 μl drops delivered using a 10-20 μl pipette with a sterile tip. Animals received one drop every two minutes to alternating nares of the nose (e.g. left, right, left, and right). One nostril was gently covered and the 6-μl drop was placed on the opening of the other nostril. Animals were left on their backs in the delivery position for an additional ten minutes after the last drop of hyaluronidase was delivered before being allowed to recover from anesthesia. Twenty minutes later, the animal was re-anesthetized for the delivery of the test article.

Treatment Delivery:

Animals were re-anesthetized as above. One million hUTC suspended in a total volume of 48 μl of CSCV4, were administered as eight 6 μl drops delivered using a 10-20 μl pipette with a sterile tip. Animals received one drop every two minutes to alternating nares of the nose. One nostril was gently covered and the 6-μl drop was placed on the opening of the other nostril. Animals were left on their backs in the delivery position for an additional ten minutes after the last drop of hUTC were delivered before being allowed to recover from anesthesia. Control animals received 48 μl of CSCV 4 without hUTC using the identical procedure. hUTC were kept on ice between doses.

Randomization and Blinding:

Animals were randomly assigned to treatment groups and the investigator, who did all of the animal surgeries and behavioral assessments, was blinded to the treatment assignment of each animal in the behavioral groups.

Behavioral Tests:

Functional activities were evaluated using limb placing and body swing behavioral tests. These tests were performed one day before surgery (Day −1), one day after surgery (Day 1, before treatments), and seven (Day 7), fourteen (Day 14), twenty-one (Day 21), twenty-eight (Day 28) and forty-one (Day 41) days after MCAo. (Day 0˜day of MCAo)

1. Limb Placing

Limb placing tests were divided into both forelimb and hindlimb tests. For the forelimb-placing test, the examiner held the rat close to a tabletop and scored the rat's ability to place the forelimb on the tabletop in response to whisker, visual, tactile, or proprioceptive stimulation. Similarly, for the hindlimb placing test, the examiner assessed the rat's ability to place the hindlimb on the tabletop in response to tactile and proprioceptive stimulation. Separate sub-scores were obtained for each mode of sensory input (half-point designations possible), and added to give total scores (for the forelimb placing test: 0=normal, 12=maximally impaired; for the hindlimb placing test: 0=normal; 6=maximally impaired).

2. Body Swing Test

The rat was held approximately one inch from the base of its tail. It was then elevated to an inch above a surface of a table. The rat was held in the vertical axis, defined as no more than 10° to either the left or the right side. A swing was recorded whenever the rat moved its head out of the vertical axis to either side. The rat must return to the vertical position for the next swing to be counted. Thirty (30) total swings were counted. A normal rat typically has an equal number of swings to either side. Following focal ischemia, the rat tends to swing to the contralateral (left) side.

Sacrifice:

At the designated time points, (e.g. 4 hours, 1 day, 3 days and 41 days after dosing), rats were anesthetized (ketamine 50-100 mg/kg/Xylazine 5-10 mg/kg), CSF was collected and placed on dry ice immediately and then stored in the −80° C. freezer before shipping. Then, the animals were sacrificed in a CO₂ chamber. Blood was collected (whole blood plus serum), and then tissues were collected, including nasal cavity, olfactory bulb, brain (cut into 10 2-mm coronal sections using a rodent brain block), lung, liver (separated into 2 tubes) and spleen. Weights were collected for all organs harvested. Below is the list of animals from which tissue was collected:

-   -   1. 1 MM cells, intranasal with HU, Day 1 post-MCAo, sacrifice         Day 1 (4 hours after dose) (N=4)     -   2. 1 MM cells Intranasal without HU, Day 1 post-MCAo, sacrifice         Day 1 (4 hours after dose) (N=4)     -   3. 1 MM cells, intranasal with HU, Day 1 post-MCAo, sacrifice         Day 2 (1 day after dose) (N=4)     -   4. 1 MM cells, intranasal without HU, Day 1 post-MCAo, sacrifice         Day 2 (1 day after dose) (N=4)     -   5. 1 MM cells, intranasal with HU, Day 1 post-MCAo, sacrifice         Day 4 (3 days after dose) (N=4)     -   6. 1 MM cells, intranasal without HU, Day 1 post-MCAo, sacrifice         Day 4 (3 days after dose) (N=4)     -   7. 1 MM cells, intranasal with HU, Day 1 post-MCAo, sacrifice         Day 42 (41 days after dose) (N=4)     -   8. 1 MM cells, intranasal without HU, Day 1 post-MCAo, sacrifice         Day 42 (41 days after dose) (N=4)         The rest of the animals were sacrificed in a CO₂ chamber without         collection of organs.

Data Analysis:

All data are expressed as mean±S.E.M. Behavioral and body weight data were analyzed by repeated measures of ANOVA (treatment X time).

For the behavioral tests, the day before treatment (stroke), Day −1 and the day of/after treatment (stroke), Day 1, are purposely not included in the analysis to ensure normal distribution of the data.

Results:

Clinical Observations and Survival: Animal #59 died under anesthesia during MCAo surgery and was replaced by #59R. Animal #19 died under anesthesia during dosing and was replaced by #19R. All of the other animals appeared normal.

Behavioral Tests: The results of the behavioral tests are shown in FIGS. 29 to 34 and Table 26-1 to 26-6. As is evident from the Figures, for forelimb and hindlimb placing, although there were no overall significant differences, there was a trend toward enhanced recovery in hUTC-treated animals. When compared to its own vehicle, the hUTC without HU group were superior to vehicle group (CSCV 4 without HU) in both forelimb and hindlimb placing tests (p<0.05). Furthermore, as is evident from the Figures for body swing, animals receiving hUTCs were superior to both of the vehicle groups after stroke (p<0.01). In addition, as is evident from FIGS. 35 and 36 (weight), there were no significant differences between groups.

TABLE 26-1 ANOVA table for Forelimb Placing Test (hUTC, 1 MM cells vs. HU, hUTC, 1 mm cells) for days after stroke Sum of Mean F- DF Squares Square Value P-Value Lambda Power Treatment 1 1.562 1.562 0.105 0.7495 0.105 0.001 Subject 18 267.485 14.860 (Group) Category 4 160.410 40.102 75.804 <0.0001 303.217 1.000 for Days after Stroke Category 4 2.200 0.550 1.040 .3929 4.159 0.305 for Days after Stroke * Treat- ment Category 72 38.090 0.529 for Days after Stroke * Subject (Group)

TABLE 26-2 ANOVA for Forelimb Placing Test (hUTC, 1 MM cells vs. HU, hUTC, 1 mm cells): Means Table for Days After Stroke Std. Std. Group Count Mean Dev. Error HU, hUTC, 1 MM Cells, Day 7 10 6.400 2.601 0.823 HU, hUTC, 1 MM Cells, Day 14 10 4.900 2.066 0.653 HU, hUTC, 1 MM Cells, Day 21 10 3.800 2.163 0.684 HU, hUTC, 1 MM Cells, Day 28 10 2.950 2.114 0.009 HU, hUTC, 1 MM Cells, Day 41 10 2.600 2.295 0.726 hUTC, 1 MM Cells, Day 7 10 6.100 .738 0.233 hUTC, 1 MM Cells, Day 14 10 5.250 1.230 0.389 hUTC, 1 MM Cells, Day 21 10 4.100 1.329 0.420 hUTC, 1 MM Cells, Day 28 10 3.550 1.554 0.491 hUTC, 1 MM Cells, Day 41 10 2.900 1.500 0.476

TABLE 26-3 ANOVA Table for Hindlimb Placing Test (hUTC, 1 MM cells vs. HU, hUTC, 1 MM cells) for days after stroke Sum of Mean F- DF Squares Square Value P-Value Lambda Power Treatment 1 0.040 0.040 0.014 .9066 0.014 0.051 Subject 18 50.900 2.828 (Group) Category 4 51.565 12.891 60.864 <0.0001 243.454 1.000 for Days after Stroke Category 4 2.085 0.521 2.461 0.0529 9.844 0.673 for Days after Stroke * Treat- ment Category 72 15.250 0.212 for Days after Stroke * Subject (Group)

TABLE 26-4 ANOVA for Hindlimb Placing Test (hUTC, 1 MM cells vs. HU, hUTC, 1 mm cells): Means Table for Days After Stroke Std. Std. Group Count Mean Dev. Error HU, hUTC, 1 MM Cells, Day 7 10 3.500 1.100 0.350 HU, hUTC, 1 MM Cells, Day 14 10 2.300 0.949 0.300 HU, hUTC, 1 MM Cells, Day 21 10 1.900 0.876 0.277 HU, hUTC, 1 MM Cells, Day 28 10 1.400 0.907 0.287 HU, hUTC, 1 MM Cells, Day 41 10 1.200 0.978 0.309 hUTC, 1 MM Cells, Day 7 10 3.000 0.577 0.183 hUTC, 1 MM Cells, Day 14 10 2.550 0.550 0.174 hUTC, 1 MM Cells, Day 21 10 1.650 0.851 0.269 hUTC, 1 MM Cells, Day 28 10 1.500 0.882 0.279 hUTC, 1 MM Cells, Day 41 10 1.400 0.738 0.233

TABLE 26-5 ANOVA Table for Body Swing Test (hUTC, 1 MM cells vs. HU, hUTC, 1 MM cells) for days after stroke Sum of Mean DF Squares Square F- Value P-Value Lambda Power Treatment 1 120.978 120.978 0.651 0.4301 0.651 0.115 Subject (Group) 18 3342.689 185.705 Category for Days after 4 7534.048 1891.012 46.064 0.0001 184.257 1.000 Stroke Category for Days after 4 70.659 17.005 0.430 0.7003 1.721 0.143 Stroke * Treatment Category for Days after Stroke * Subject 72 2955.716 41.052 (Group)

TABLE 26-6 ANOVA for Body Swing Test (hUTC, 1 MM cells vs. HU, hUTC, 1 mm cells): Means Table for Days After Stroke Std. Std. Group Count Mean Dev. Error HU, hUTC, 1 MM Cells, Day 7 10 23.334 9.686 3.003 HU, hUTC, 1 MM Cells, Day 14 10 35.000 7.738 2.447 HU, hUTC, 1 MM Cells, Day 21 10 40.999 8.172 2.584 HU, hUTC, 1 MM Cells, Day 28 10 44.334 8.895 2.813 HU, hUTC, 1 MM Cells, Day 41 10 45.332 8.489 2.685 hUTC, 1 MM Cells, Day 7 10 18.001 10.910 3.450 hUTC, 1 MM Cells, Day 14 10 34.007 5.710 1.8a3 hUTC, 1 MM Cells, Day 21 10 38.999 8.468 2.678 hUTC, 1 MM Cells, Day 28 10 43.000 6.749 2.134 hUTC, 1 MM Cells, Day 41 10 43.333 7.698 2.434

No CSF was collected from #12 due to technical difficulty (no space between brain and dura).

CONCLUSION

Permanent right MCAo by the Tamura method was made in mature male Sprague-Dawley rats, resulting in focal right cerebral infarction. Human UTCs (1 MM) were administered intranasally with or without hyaluronidase (HU) pretreatment, at 1 day after MCAo, compared to appropriate controls. Behavioral assessments of sensorimotor function were made at 1 day before MCAo, 1 day after MCAo, and at 7, 14, 21, 28 and 41 days after MCAo.

Human UTCs, given at 1 day after stroke, with or without HU, enhanced sensorimotor recovery compared to respective vehicle-treated controls on behavioral tests, especially on the body swing test. In this study, it was demonstrated that hUTC can improve neurological recovery in rats when delivered intranasally.

The present invention is not limited to the embodiments described and exemplified above. It is capable of variation and modification within the scope of the appended claims. 

1. A method of treating stroke in a patient comprising intranasally administering to the patient umbilical cord tissue-derived cells in an amount effective to treat stroke, wherein the cells are isolated from mammalian umbilical cord tissue substantially free of blood or are expanded in culture from a cell isolated from mammalian umbilical cord tissue substantially free of blood, wherein the cells are capable of self-renewal and expansion in culture, and wherein the cells do not produce CD117.
 2. The method of claim 1, wherein the umbilical cord tissue-derived cells do not express hTERT or telomerase.
 3. The method of claim 1, wherein the cells further have one or more the following characteristics: (a) express each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C; (b) do not express any of CD31, CD34, CD45, CD80, CD86, CD 117, CD141, CD178, B7-H2, HLA-G, or HLA-DR,DP,DQ; (c) secrete at least one of MCP-1, IL-6, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, I309, MDC, and TIMP1 (d) do not secrete at least one of TGF-beta2, ANG2, PDGFbb, MIP1a, and VEGF, as detected by ELISA; and (e) increased expression of interleukin-8; reticulon 1; and chemokine receptor ligand (C-X-C motif) ligand 3, relative to that of a human cell which is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell.
 4. The method of claim 1, wherein the stroke is ischemic stroke.
 5. The method of claim 1, wherein the stroke is hemorrhagic stroke.
 6. The method of claim 1, wherein the method further comprises treating the patient with hyaluronidase prior to intranasally administering the cells to the patients.
 7. The method of claim 1, wherein the method further comprises administering an immunosuppressive agent.
 8. The method of claim 7, wherein the immunosuppressive agent is cyclosporine.
 9. The method of claim 1, wherein the cells are administered with at least one other cell type of an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell, genetically engineered cell, or other multipotent or pluripotent stem cell.
 10. The method of claim 9, wherein the at least one other cell type is administered simultaneously with, or before, or after, the umbilical-derived cells.
 11. The method of claim 1, wherein the umbilical cord tissue-derived cells are genetically engineered to produce a gene product that promotes treatment of stroke.
 12. The method of claim 1, wherein the cells exert a trophic effect on the nervous system of the patient.
 13. The method of claim 1, wherein the umbilical cord tissue-derived cells are induced in vitro to differentiate into a neural cell line prior to administration.
 14. A method of treating stroke in a patient comprising intranasally administering to the patient a pharmaceutical composition comprising umbilical cord tissue-derived cells in an amount effective to treat stroke, wherein the cells are isolated from mammalian umbilical cord tissue substantially free of blood or are expanded in culture from a cell isolated from mammalian umbilical cord tissue substantially free of blood, wherein the cells are capable of self-renewal and expansion in culture, and wherein the cells do not produce CD117.
 15. The method of claim 14, wherein the umbilical cord tissue-derived cells do not express hTERT or telomerase.
 16. The method of claim 14, wherein the cells further have one or more the following characteristics: (a) express each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C; (b) do not express any of CD31, CD34, CD45, CD80, CD86, CD 117, CD141, CD178, B7-H2, HLA-G, or HLA-DR,DP,DQ; (c) secrete at least one of MCP-1, IL-6, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, I309, MDC, and TIMP1 (d) do not secrete at least one of TGF-beta2, ANG2, PDGFbb, MIP1a, and VEGF, as detected by ELISA; and (e) increased expression of interleukin-8; reticulon 1; and chemokine receptor ligand (C-X-C motif) ligand 3, relative to that of a human cell which is a fibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell.
 17. The method of claim 14, wherein the stroke is ischemic stroke.
 18. The method of claim 14, wherein the stroke is hemorrhagic stroke.
 19. The method of claim 14, wherein the method further comprises treating the patient with hyaluronidase prior to intranasally administering the pharmaceutical compositions to the patients.
 20. The method of claim 14, wherein the pharmaceutical composition further comprises hyaluronidase.
 21. The method of claim 14, wherein the pharmaceutical composition further comprises an immunosuppressive agent.
 22. The method of claim 21, wherein the immunosuppressive agent is cyclosporine.
 23. The method of claim 14, wherein the pharmaceutical composition is administered as a spray, aerosol, gel, solution, emulsion, or suspension.
 24. The method of claim 23, wherein the pharmaceutical composition is administered directly to the upper airways.
 25. The method of claim 14, wherein the pharmaceutical composition is administered directly to the paranasal sinuses.
 26. The method of claim 14, wherein the pharmaceutical composition is administered via a microcatheter.
 27. The method of claim 14, wherein the pharmaceutical composition further comprises at least one other cell type.
 28. The method of claim 27, wherein the at least one other cell type is an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell, genetically engineered cell, or other multipotent or pluripotent stem cell.
 29. The method of claim 14, wherein the umbilical cord tissue-derived cells are genetically engineered to produce a gene product that promotes treatment of stroke.
 30. The method of claim 14, wherein the pharmaceutical composition exerts a trophic effect on the nervous system of the patient.
 31. The method of claim 14, wherein the umbilical cord tissue-derived cells are induced in vitro to differentiate into a neural cell line prior to administration. 